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Iron is required by virtually all organisms. Many microorganisms, both eukaryotes and prokaryotes, synthesize specific iron-chelating compounds called siderophores (also known as ironophores) which are extracellular (i.e., exported outside of the cell) and are involved in the solubilization and transport of iron compounds into the cell. (Siderophores may perform additional functions in the metabolism of microorganisms.) Microbially-produced siderophores act as growth factors (see Appendix D) for organisms which cannot synthesize them. Thus, in the mixed microbial populations of natural environments, siderophores essential to the growth of certain organisms are supplied by the excessive secretions of these substances by other organisms which can synthesize them. This experiment will do nothing more than demonstrate the requirement of an exogenously-supplied siderophore for growth of a siderophore auxotroph and the ability of the auxotroph to be supplied the siderophore artificially and by certain other microorganisms.

Period 1

Materials

Saline suspension of cells of the siderophore auxotroph, Arthrobacter flavescens (strain

Broth cultures of Bacillus subtilis, Streptomyces griseus and Rhodotorula rosei (a yeast)

3 plates of Brain Heart Infusion (BHI) Agar plates 3 sterile swabs

Sterile paper disc saturated with a solution of Deferrioxamine B (5µg/ml), a commercially- available siderophore obtained from a species of Streptomyces and normally used in the treatment of chronic iron storage disease.

Procedure

1. Inoculate each plate of BHI Agar with the Arthrobacter flavescens suspension by means of the sterile swabs. Make sure each plate is completely and evenly covered with the inoculum. Discard the swabs into the disinfectant.
2. At the edge of one of the plates, aseptically (with flame-sterilized forceps) place a Deferrioxamine-impregnated disc. At the opposite edge of the same plate, spot-inoculate (just a dab with your loop - not a streak) the Bacillus subtilis culture.
3. On the second plate, at opposite edges, spot-inoculate Streptomyces griseus and Rhodotorula rosei.
4. Leave the third plate as is, with no further treatment. This is the control plate which will show how poorly the A. flavescens grows (if it grows at all) on a medium not supplemented with the required growth factor (i.e., the siderophore).
5. Incubate the plates at 30 °C for 3 or more days.

Period 2

Procedure

1. Observe the plates for the presence or absence of satellite growth of A. flavescens around the inoculation spots and the Deferrioxamine disc. Which organisms can provide the siderophore? For the organism which does not appear to provide a siderophore which the A. flavescens can utilize, would you expect that organism to be producing one anyway?

Example of phenotypic variation

Genotype is defined as the entire array of genes possessed by a cell, i.e., the sum of the genetic constitution of the organism, a blueprint in code. The characteristics of an organism which are based on the genotype but expressed within a given environment make up the phenotype of the organism. Thus, the genotype represents the potential of the organism, and the phenotype describes what the organism actually is and does. For example, in some species of bacteria, the production of a pigment is a phenotypic manifestation of the genotype, and the degree of actual pigment production may be influenced by temperature or nutritional variations. As another example, the ability to produce a siderophore is a genetically-controlled characteristic, but it may or may not be expressed depending on the amount of iron in the environment. When iron is in low levels, the organism will produce much more of the siderophore in order to scavenge the iron.

In this experiment, we will utilize an organism, Pseudomonas fluorescens, which produces a pigmented siderophore (fluorescein) which is easy to detect with the naked eye if it is produced in sufficient quantity. It also glows (fluoresces) when illuminated with ultraviolet light. When inoculated onto two media which differ in the concentration of iron compounds, we will see how the same organism appears differently on each medium, an example of phenotypic variation, a difference in fluorescein synthesis.

Since phenotypic characteristics are generally used to differentiate between microorganisms, it is important that all tests be done in a standardized set of conditions (medium, temperature, time, etc.).

SPECIAL SAFETY PRECAUTION: BE CAREFUL WHEN USING THE ULTRAVIOLET LIGHT. USE THE SAFETY GLASSES PROVIDED. Ultraviolet light damages the retina of the eye, and prolonged exposure to the light reflecting off the plates can also cause damage.

Period 1

Materials

Broth culture of Pseudomonas fluorescens

1 plate each of Nutrient Agar and Pseudomonas Agar-F

Procedure

1. With a single streak with the loop, inoculate the culture onto each of the plates.
2. Incubate the plates at 30 °C for 2 or more days.

Period 2

Materials

Ultraviolet lights set up in a dark room

Procedure

1. Observe the plates for production of fluorescein by the culture. This usually appears as a yellowish-green color diffused into the medium.
2. To observe the fluorescence of the pigment (hence its name), take the plates to the dark room.

Remove the covers and expose the cultures to the ultraviolet lamp. SEE THE SAFETY PRE-CAUTION ABOVE REGARDING ULTRAVIOLET LIGHT. The glowing, yellow-green fluorescence of the pigment is not itself ultraviolet light, but light within the normal, visible range. Which of the two media apparently contains more iron? (Note that iron is a trace element in each medium; this was also the case in the BHI Agar used in Experiment 2.

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