Note
Studied by 25 peopleHost Basics_ Gordonia terrae
An Introduction to the Host Bacteria, Gordonia terrae
As a network of researchers, SEA-PHAGES explores the genetic diversity and relationships of populations of bacteriophages that infect Actinobacteria, incluidng the bacterium Gordonia terrae. There are many different strains of Gordonia terrae (isolated from different samples). The Gordonia terrae strain widely used in SEA-PHAGES is called Gordonia terrae CAG3 that was originally isolated from oil-contaminated soil in China. If you intend to isolate phages using this bacterium, then this will be your ‘host’ for phage isolation.
G. terrae. is one of over 19 species that make up the genus Gordonia, classified within the phylum Actinobacteria. G. terrae. is a gram-positive soil organism, whose relatives are well equipped to breakdown various hydrocarbons. This ability makes Gordonia attractive bacteria for use in bioremediation (i. e. the breakdown of pollutants) and in industrial biotechnology. For example, Gordonia sp. MTCC 4818 is able to breakdown phthalic acid esters, which can act as hormones that disrupt normal body function. Another Gordonia species, CYSK-1, is able to remove sulfur from fossil fuels, which will allow for the production of cleaner fuels, such as ultra-low-sulfur diesel.
G. terrae. will grow and divide on many nutrient-rich media. In your research, you will grow G. terrae on PYCa media and at 30 ˚C. Under these conditions G. terrae grows rapidly, dividing approximately once every 2 - 3 hours. This means that it typically takes about 3 days for an individual cell to form a colony on an agar plate. However, when preparing a bacterial lawn, you can start by adding many
bacterial cells (e. g. 107 or more) so that a lush lawn grows within 2 – 3 days. For reproducibility of experiments, it is important that cultures used for a given set of experiments be prepared at the same temperature.
G. terrae colonies are tan to orange in color, round, rough and dry in texture (Figure 4.0-4). When incubated for prolonged periods, the color of the colonies intensifies. A liquid culture of G. terrae inoculated from a single colony will take 2 – 3 days to form a saturated liquid culture, exhibiting a tan to orange color. The strain you will work with, G. terrae CAG3, is not inhibited by the antifungal cyclohexamide, or the antibiotic ampicillin at low concentrations. Therefore, this can be added to
the growth media to prevent other microorganisms from growing in your cultures.
On average, 33 % of attempts to isolate phage from soil by enrichment with Gordonia terrae yield phage.
 |
|---|
Figure 4.0-4. Gordonia terrae growing on an agar plate. |
If you are using this bacterium as your host for phage isolation, refer back to the list below for the specific growth and culture requirements for your experiments.
Growth media: PYCa, supplemented with Tween80 (see instructions for culturing bacteria that require Tween80)
Growth temperature: 30 ˚C
Antimicrobials: cycloheximide (10 μg/ml), ampicillin (optional, and up to 10 mg/ml)
Phage Buffer: 10 mM Tris (pH 7.5), 10 mM MgSO4, 68 mM NaCl, 1 mM CaCl2, (10% glycerol, optional)
Restriction Enzymes: BamHI, ClaI, EcoRI, HaeIII, HindIII, MseI.
Note: Isoschizomers, which are different restriction enzymes that recognize the same DNA sequence, may be used in place of any of the enzymes listed above.