Question

Part II ---- Suspicious Minds

Your direct microscopic observation of microorganisms in the soil samples has sparked your boss’s interest. He is eager to determine what type of microorganism(s) is present: eukaryotic or prokaryotic. Gram-positive or Gram-negative, or maybe even something new, never before seen on Earth. He sends a sample of the soil

off to a biochemistry laboratory for direct analysis.

You are equally interested in the nature of the microbes, but instead of directly analyzing the soil, you first isolate a pure culture of a microorganism that you demonstrate has the ability to degrade polyurethane. You

send a sample of this pure culture to the same biochemistry laboratory for analysis.

Later, you receive the results of the analysis of your boss’s sample and your pure sample

Table 1
Test	Boss’s sample	Your sample
80 S ribosome	+	–
70 S ribosome	+	+
Circular DNA	+	+
Linear DNA	+	–
RNA	+	+
Phospholipid membranes containing electron transport proteins	+	+
LPS	+	+
Lipoteichoic acid	+	–
Flagellar basal body proteins	+	+
Pilus proteins	+	+
Nuclear pore proteins	+	–
Histone proteins	+	–

“I’m not sure what’s wrong with your sample, but my results prove that we are dealing with a new kind of life form here….I’m calling it the preuk-aryote” because it has components characteristic of both prokaryotes

and eukaryotes. It’s time for a press conference!” boasts your boss.

Later on, as you are getting ready to head home after a long day in the lab, you hear your boss bellow, “What

the H-E-double hockey sticks is going on here!”

You ask him what happened.

“This morning I put a few thousand cells from your pure culture of Extraterrestrial PolyUrethane-Degrading Microbe (EPTUM) onto two slides in some water, but then I had to go to that press conference, and I didn’t have enough time to look at the cells carefully except to notice that they were uniformly distributed under the coverslip. I didn’t want the slides to dry out so I sealed the edges of the coverslips. On this slide I used a rubber gasket to make the seal, and on this slide I used a Lycra gasket. Now look at the cell distribution! On the rubber-sealed slide, the cells are still uniformly distributed, but on the Lycra-sealed slide all the cells have congregated around the edge of the coverslip. Look….they are all over at the edges; none are left in the middle part of the slide. Could somebody have come in here and moved all those EPTUM cells over to the edges? But who? Maybe someone small with really tiny tweezers. Did you see anyone like that lurking around this scope? Nah….I need to get a grip on reality. No tweezers could be that small.”

Questions

3.  What technique is used to isolate a pure bacterial culture?

4. If your goal is to characterize the ETPUM, whose results are more informative: yours or your boss’s?  

    Why? Does its biological composition most closely resemble that of a prokaryote or a eukaryote? Gram-

    positive or Gram-negative? Do you agree with your boss’s conclusion that the ETPUM is a prokaryotic-

    eukaryotic hybrid? Why or why not?

5. Come up with at least two possible alternative explanations for the “amazing” redistribution of the

    ETPUM on the Lycra-sealed slide. Both of your explanations should consider how the microbes “sensed”

    the presence of polyurethane. One of your answers should not involve flagella.

Part III----All Shook Up

You have found media that support growth of pure cultures of EPTUM in your laboratory. The recipes for these media are shown below:

Table 2
Medium #1 (per liter H2O)	Medium #2 (per liter H2O)
5 g yeast extract	10.5 g K2HPO4
20 g tryptone extract	4.5 g KH2PO4
0.5 g NaCl	1 g MgSO4
3.6 g glucose	10 g polyurethane

Growth in these media:

Table 3
Growth	Medium #1	Medium #2
ETPUM growth---aerobic	+	+
ETPUM growth---anaerobic	+	–
E. coli growth----aerobic	+	–
E. coli growth----anaerobic	+	–

You are excited because, in Medium #2, ETPUM utilizes polyurethane as its energy source and its sole source of carbon and nitrogen, a finding that raises the possibility that ETPUM could be a useful tool for bioremediation of polyurethane-containing wastes (in landfills, etc.). You have also made some progress in characterizing the central metabolic pathways and related  biochemical activities of ETPUM. In particular you have discovered that:

• ETPUM secretes an enzyme (polyurethanase) that catalyzes degradation of polyurethane, generating citric acid (citrate) as a product.
• The cytoplasmic membrane of ETPUM contains an ABC transport system capable of transporting citrate across the membrane at the expense of 4 ATP molecules (hydrolyzed to form ADP and phosphate) per molecule of citrate transported.
• The cytoplasm of this organism contains all of the enzymes required for glycolysis, and for the TCA cycle.
• The cytoplasmic membrane of ETPUM contains proteins that form a functional electron-transport pathway (that uses O₂ as the terminal electron acceptor)

Questions

6. Which medium would you consider to be “complex” and which “defined”?  Which is “rich” and which is

     “minimal”?  Explain your answers.

7. Given that polyurethane is a huge polymer (MW>>100,000 Daltons). Why is it important that the

    polyurethanase is a secreted enzyme? If we assume that the polyurethane is the source of energy for the

    organism, how can material (carbon atoms) from it find its way into the central metabolic pathways of this

    microbe? What is the “entry point”? What happens after its entry into the metabolic pathway?

8. Why does growth of EPTUM in Medium #2 require oxygen? Think about this in terms of how EPTUM

    can generate a net gain of in ATP processing polyurethane. Remember that the degradation of

    polyurethane by polyurethanase does not expend ATP. In order to answer this question, address each of

    the following questions in your answer:

    a. Is there a net gain or loss of ATP during the transport of citrate?

    b. Consider the ATPs that can be generated via substrate-level phosphorylation. Will glycolysis be useful  

        for generating any ATPs during growth on polyurethane? How many ATPs can be generated via TCA

        (i.e via substrate level phosphorylation)? Is this enough to support growth (is there a net positive in the

        ATP tally)?

    c. Now consider how else ETPUM can generate ATPs (if not by substrate-level-phosphorylation). Can

        this process generate a net positive in the ATP tally

    d. Now explain the importance of oxygen as relates to the ATP tally.

Answer 1

3. The streak plate method: The streak plate method is commonly used to isolate pure bacterial cultures in a special media with specific physical or chemical agents that allow the growth of selected bactria. Using an inoculation loop, a small amount of bacterial culture is streaked on the surface of an agar plate and we will get pure isolated colonies.

4. In the case of ETPUM  (Extraterrestrial PolyUrethane-Degrading Microbe) my results are more informative than my boss's results because he used a mixed culture of bacterial sample which contain more than one organism.

ETPUM is a gram -ve bacteria. No, I do not agree with my boss’s conclusion that the ETPUM is a prokaryotic-eukaryotic hybrid because his sample is a mixed culture contain more than one bacteria. So the result shares both prokaryotic and eukaryotic features. My culture is a pure sample and it shows prokaryotic features of bacteria. So my results indicates that ETPUM is a prokaryote.

5. The “amazing” redistribution of the ETPUM on the Lycra-sealed slide is due to the motility of bacteria. Bacterial cells uses flagella for their movement. All the bacterial cells on the Lycra-sealed slide have congregated around the edge of the coverslip. Bacterial cells also have the ability to move by taxis (in response to a stimulus such as light). Bacterial cells responses to any stimulus such as light, temperature or presence of food via taxis. This is the way the microbes “sensed” the presence of polyurethane.

6. Complex medium - A complex media contain complex materials of biological origin which means the composition of complex material (eg; milk, blood, yeast extract and beef extract) is not known. Eg; Nutrient Agar media (peptones - 5 g, beef or yeast extract - 3 g and agar -15 g per 1 liter of water).

Defined medium - A defined medium contain. known the exact amount of chemical composition. eg; example peptone, it is a source of amino acids, nitrogen (N), sulphur (S), and phosphorus (P).  

Minimal medium - In a minimal medium contain minimum nutrients for the growth of microorganisms. eg; carbon source, nitrogen source, salts and trace metals. eg; nutrient media.

Rich medium - A rich medium contains a variety of nutrients. eg; blood agar.

7. Polyurethanase is a secreted enzyme by the microbes. It is used to break down polyurethane. Polyurethane is an important enery source for the ETPUM, so it needs to be broken down. The material (carbon atoms) from Polyurethane enters through the membrane by diffusion and to the central metabolic pathways by catabolism. EPTUM degrades polyurethane to citrate.

8. For the ATP production the growth of EPTUM in Medium #2 require more oxygen. The polyurethanase breaks polyurethane as citric acid which passes through the cytoplasm using BAC transport system (a structure with two transmembrabe domains associated with two associated hydrophilic ATPase).

a. There is a net of 2 ATP during the transport of citrate.

b. 14 ATPmolecule produced by  TCA cycle

c. Two ATP molecules are required to start glycolysis and four are generated by substrate-level phosphorylation.

d.  Aerobic respiration - 38 ATP, anaerobic respiration - 2 ATP.