Microbiology Concepts of Culture,
Growth, and Metabolism
Richard C. Stewart, Ann C. Smith and Patricia A. Shields
Department of Cell Biology and Molecular Genetics
University of Maryland—College Park, MD Part I—Return to Sender
Fresh out of college, with your degree in microbiology, you have landed your “ﬁrst real job” as a scientist with
DuPunt, a company that specializes in development and production of polyurethane derivatives (specialized
plastics). You (and your boss) are not quite sure why DuPunt has a microbiologist on staﬀ, but you are both
about to ﬁnd out why the company desperately needs one now. Your boss has called you into his oﬃce. “Read
this article!” he says, pushing the front page of a major national newspaper across his desk to you.
ELVIS NAKED, SKINLESS UPON RETURN FROM OUTERSPACE!
The recent return to Earth of the unmanned exploration probe ELV from the
Nearby Previously Invisible Planet (NPIP) has provided scientists with important
information about conditions on the surface of this recently discovered planet.
Although physicists have made great advances in understanding how NPIP recently
was “uncloaked,” perhaps the most interesting discoveries are yet to come as
biological scientists begin to analyze the soil samples collected by the probe’s
Extraterrestrial Landing Vehicle Integrated Sampler (ELVIS). Designed to gather
samples and maintain them in their normal atmospheric and temperature conditions,
this collection unit is a sophisticated robot and is the most expensive component of
the ELV probe. Inspired by the acronym for the unit (ELVIS), workers constructed
this robot to resemble music legend Elvis Presley, and even fashioned a white
Spandex jumpsuit to clothe it.
These design features have proven to be a public relations coup, endearing the 2foot-tall robot to the public and making it the unofﬁcial mascot of the NASA space
exploration program. This “human connection” has been instrumental in convincing
Congress to provide the necessary funding for the ELV and many related space
However, the successful completion of the ELV mission has generated a mystery,
one that has led to accusations of contractors providing substandard materials
(in particular defective plastics) used in the ELV or of someone intentionally
sabotaging the plastic components of the ELV in an attempt to embarrass the U.S.
space exploration program. “ELVIS Meltdown!” by Stewart, Smith and Shields Page Upon its return to Earth, the ELV capsule was opened to allow scientists to recover the
soil samples. This was done as part of a special, televised “welcome home” ceremony,
in which ELVIS was supposed to “dance” down the ELV exit ramp and speak his
trademark words, “Thank you … thank you very much for supporting this critical
space exploration mission.”
NASA scientists and on-lookers at the ceremony were shocked to ﬁnd that ELVIS’s
jumpsuit had been reduced to a slimy puddle. Even more distressing was the
deterioration of ELVIS’s “skin” (a version of Lycra specially developed to resemble
human skin). This too was reduced to a slimy residue that dripped from the metal
“skeleton” of the ELVIS unit. The deterioration of the plastic components of ELVIS
ruined what organizers had planned to be a touching ceremony at the mission’s
completion. ELVIS’s exit from the otherwise intact spacecraft was met by gasps and
screams from the gathered audience. “It was a terrible sight!” said one member of
the audience. “We expected to see the King, but we saw a horrible mess, a grotesque
parody of Elvis. Without his plastics lips, I couldn’t understand a word he said … and
the smell was horrible!” said one NASA ofﬁcial who wished to remain anonymous.
Television viewers were spared much of the trauma of these sights as networks quickly
switched to new episodes of Sponge Bob Squarepants in which a cartoon version of
the ELVIS unit was featured. An investigation is underway. Stiﬂing your initial reaction (“Oh yeah, new Sponge Bob!”) you manage to mumble, “What a tragedy!”
“Yes. Yes. And this could take an ugly turn for DuPunt!” your boss says. “I’m not sure what caused this mess,
but I do know a couple of things that didn’t make it into that news article: () the only plastics showing
damage in the ELV were polyurethanes; and () our company provided those polyurethane products to
NASA at a cost of $,,. We’re in big trouble if we can’t prove that something from that planet is
responsible for destroying ELVIS.”
He continues, “The polyurethane products we provided were ﬁrst-rate. We didn’t cut any corners with this
stuﬀ. Products from the same batches of polyurethane have been into outer space before, and they returned
just ﬁne. There must be some explanation other than our incompetence. This is where you come in. I need
you to ﬁnd that explanation!”
“Why me?” you ask.
“Because of the stink!” your boss answers. “Some of the scientists present at the ELVIS disaster said the smell
reminded them of an old fermenter or an autoclave. Those are microbiology terms, aren’t they?” says your
boss. “Those comments tell me that this whole stinking mess might be caused by microorganisms—you
know, bacteria, fungi, viruses, germs … something like that. Get right to work on this! You and I will have
to work closely on this, you know. I’ll handle all the communications with the press, and you handle the
science. Just make sure that you explain everything to me so that I can speak about it to the press without
making a fool of myself and DuPunt!” “ELVIS Meltdown!” by Stewart, Smith and Shields Page Okay. You are a trained scientist … you can do this! What do scientists do? They answer questions by
testing speciﬁc hypotheses. As a microbiologist at DuPunt you must determine what has happened to the
polyurethane. Here is your hypothesis:
The degradation of polyurethane products was caused by a microorganism or microorganisms present in
the soil samples collected by ELVIS. Questions
. Using light microscopy, you examine the soil samples and the “goo” from the degraded polyurethane.
Will this approach allow you to observe all microorganisms present in the samples? Why or why not?
What are the limitations of this approach?
. You use phase contrast microscopy to observe a wet mount of a soil sample (the ﬁrst picture below) and
a “goo” sample (the second image below) from the ELVIS. In what ways are the potential ET microbes
similar to microbes previously characterized on Earth? In what ways are they diﬀerent? How could
you determine whether the microbes present in the soil or goo samples are phylogenetically similar or
distant from known microorganisms on Earth? . Your boss has done a little reading about microorganisms, but he ﬁnds it all pretty complicated. “It’s
like a foreign language!” he complains. “I have to face the press to explain our idea that microbes
might be responsible for all the damage to ELVIS. I think that it will help my press conference
presentation a lot if I can use some visual aids. What I want to do is explain to my audience what
bacteria look like. You know… the functional architecture of bacteria and how they might be able to
degrade polyurethane. I think that eukaryotes might be too complicated for this audience, so I just
want to show them what Gram-negative bacteria look like in a schematic diagram. I’ve put together
this diagram of a typical Gram-negative cell. Take a look at it and make any corrections you think
are necessary. Notice that I not only labeled the features, I also indicated the major biochemical
composition and function(s) of each main feature. Oh yeah … this ﬁgure will probably make it into
lots of newspapers, magazines, and web sites, so it needs to be scientiﬁcally correct. We wouldn’t want
to make DuPunt look stupid, would we? I’ve already done most of the work. Just proofread it and
make any necessary corrections.” (See Figure 3 next page.) “ELVIS Meltdown!” by Stewart, Smith and Shields Page “ELVIS Meltdown!” by Stewart, Smith and Shields Page 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.
Test Boss’s sample Your sample S ribosomes + − S ribosomes + + Circular DNA + + Linear DNA + − RNA + + Phospholipid membranes containing
electron transport proteins + + Peptidoglycan + + 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 (ETPUM) 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
“ELVIS Meltdown!” by Stewart, Smith and Shields Page 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 ETPUM 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 here. No tweezers could be that small.” Questions
. How would you go about isolating your pure culture?
. If your goal is to characterize the ETPUM, whose results are more informative: yours or your
boss’s? Why? What do your results indicate about the nature of this microbe? Does its biochemical
composition most closely resemble that of a prokaryote or a eukaryote? Gram-positive or Gramnegative? Do you agree with your boss’s conclusion that the ETPUM is a prokaryotic-eukaryotic
hybrid? Why or why not?
. 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. “ELVIS Meltdown!” by Stewart, Smith and Shields Page Part III—All Shook Up
You have found media that support growth of pure cultures of ETPUM in your laboratory. The recipes for
these media are shown below:
Medium #1 Medium #2 g yeast extract . g KHPO g tryptone extract . g KHPO . g NaCl g MgSO . g glucose g polyurethane l H l H Growth in these media:
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 #, 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 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 O as the terminal electron acceptor). Questions
. Which medium would you consider to be “complex” and which “deﬁned”? Which is “rich” and which
is “minimal”? Explain your answers.
. Given that polyurethane is a huge polymer (MW >>, 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
“ELVIS Meltdown!” by Stewart, Smith and Shields Page 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?
. Why does growth of ETPUM in Medium # require oxygen? Think about this in terms of how
ETPUM can generate a net gain in ATP by 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 the 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? 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. “ELVIS Meltdown!” by Stewart, Smith and Shields Page Part IV—A Little Less Conversation
. At a press conference announcing your company’s successful isolation and characterization and
cultivation of ETPUM, a reporter raises an important question: “How do you know that this microbe
actually came from NPIP (the Nearby Previously Invisible Planet) and not from Earth? Could this
microbe really be an Earth microbe that was present in/on ELVIS before it was launched into space?”
“That’s impossible,” your boss answers. “Prior to launch, we wiped down the entire ELV and ELVIS
with the disinfectants ethanol and triclosan. In addition we used plastics impregnated with disinfectant
chlorhexidine. I had our chemists check! These chemicals do all kinds of nasty things to cells: coagulate
proteins and destroy membranes. No microbes could have survived that.”
What do you think? Were these treatments adequate to rule out the possibility raised by the reporter?
. You are a consultant for a second ELVIS mission to NPIP. Describe what physical and/or chemical
treatments you would require prior to liftoﬀ to minimize the opportunity for contamination of the
ELV (the landing module of the spacecraft) and ELVIS (a new version of the robot) by Earth microbes.
HINT: Keep in mind the following: ELVIS’s skin cannot withstand temperatures above °C; the ELV
itself is approximately the size of a minivan and includes many metal parts. Use scientiﬁc terminology
as you discuss this answer: e.g., are you trying to achieve disinfection or sterilization? Are you
recommending use of antiseptics or disinfectants? Specify how each of your treatments would achieve
killing of microbes.
. In your opinion, is EPTUM an earthly isolate or an extraterrestrial isolate? Some things to think about:
Have bacteria been isolated from outer space to date? What is this ﬁeld of study? What agencies fund
this ﬁeld? What is “Planetary Protection”? Are there earthly microbes that have not yet been isolated
and grown in culture? If so, what is the predicted percentage? Are there methods to study microbes that
cannot be cultured? Title block credit: Photo based on a licensed original ©Lise Gagne/iStockPhoto.com.
Case copyright © by the National Center for Case Study Teaching in Science. Originally published November , at
http://www.sciencecases.org/elvis/case.asp. Please see our usage guidelines, which outline our policy concerning permissible
reproduction of this work.
“ELVIS Meltdown!” by Stewart, Smith and Shields Page
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