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Topic – Batch Culture Growth
Given a set for the growth of a bacterial culture,
(6) A. Make a graph of log10OD as a function of time.
(8) B. Calculate , g, and k.
2) C. Indicate in which parts of the graph where primary and secondary metabolism are
occurring
(1) Which of the following phases of growth of a batch culture of microorganisms is
characterized by ribosome synthesis?
a. lag
b. exponential
c. stationary
d. death
(1) Which of the following inoculums would show the shortest lag period when
transferred to glucose minimal media?
a.
b.
c.
d.
a stationary-phase culture grown on TSB
a stationary-phase culture grown on glucose minimal media
an exponential-phase culture grown on TSB
a exponential-phase culture grown on glucose minimal media
(1) Which of the following would be major cell activities during secondary metabolism
a. antibiotic production
b. competence
c. pigment production
d. all of the above
e. none of the above
(1) Which of the following would be major cell activities during primary metabolism
a. fermentation
b. aerobic respiration
c. anaerobic respiration
d. all of the above
6. Which of the following phases of growth of a batch culture of microorganisms is
characterized by endogenous respiration
a. lag
b. exponential
c. stationary
d. death
Topic – Continuous Culture
In a glucose-limited chemostat, which of these mathematical statements is incorrect?
a. D = 
b. S < Sr
c. D = V / f
d. dX/dt = X - DX
Which of the following nutrients is limiting in an aquatic environment?
a. Nitrogen
b. Carbon
c. Phosphorous
d. Hydrogen
Which of the following nutrients is limiting in a terrestrial environment?
a. Nitrogen
b. Carbon
c. Oxygen
d. Hydrogen
(16 points) Continuous culture problem
Consider two species of bacteria that have the following growth parameters with
glucose as the limiting nutrient. Organism A has a Ks for glucose of 1.0 mM, and max of
2.5 hr –1. Organism B has a Ks for glucose of 0.2 mM, and max of 1.0 hr –1.
A. On the piece of graph paper provided, draw a line for each organism indicating the
growth rate as a function of substrate concentration. Indicate the Ks and max for each.
Make the y-axis 0-3.0 hr –1, and the x-axis 0 – 2.0 mM.
B. Calculate the instantaneous growth rate, μ, (to 3 significant figures) of each organism
after inoculation into APT broth (10.0 g glucose per liter) and glucose minimal medium
(0.100 % glucose or 1.00 g glucose per liter).
C. Assume that APT medium is in the reservoir of the chemostat, and glucose is the
limiting factor for growth. What would be the steady state glucose concentration, S, in
chemostats operated at a dilution rate of 0.5 hr –1 for each organism?
D. Again assuming APT is in the reservoir, and if the yield of bacteria (Yglucose, g dry cell
weight per g glucose) is 0.28, what will the steady state mass of bacteria, B, at D=0.5hr–1
for each microorganism? (Don’t assume all the substrate is consumed!)
Phosphorous is typically the limiting nutrient in
a. soils
c. infections
b. aquatic environments
d. anaerobic environments
In a glucose-limited chemostat, which of these mathematical statements is incorrect?
a. D = 
b. S > Sr
c. D = f / V
d. dX/dt = X - DX
Topic Anabolism
Nitrogen is incorporated into cell material when ammonia combines with which of the
following cell intermediates to form glutamate via the enzyme glutamate dehydrogenase.
a.
b.
c.
d.
-ketoglutarate
succinyl-CoA
oxalacetate
erythrose-4-phosphate
Topic - Pathways
Auxotrophic mutants blocked in the first step of an amino acid biosynthesis pathway
a. are easily cross-fed
b. do not overproduce any intermediates
c. are wild type
d. must be supplied the amino acid in question to survive
(10 points) Describe the steps necessary to isolate an Arg- mutant blocked in the
synthesis of arginine, but not the synthesis of citrulline by considering the following
questions. Give the arginine biosynthetic pathway starting from glutamate. What would
the frequency of such a mutant be? How would you induce mutation? How could you
increase the frequency of this auxotroph in the population? Describe the process in
detail. Describe the composition of agar media used to isolate the mutant.
(10 points) Consider the following synthesis pathway
Precursor  Intermediate 1  Intermediate 2  Amino acid X
If the first step is mediated by enzyme A, the second by enzyme B, and the third by
enzyme C, and the pathway is regulated by simple feedback inhibition,
A. Predict the outcome of a cross feeding experiment using A-, B-, and C- mutants.
Shade the areas where growth would occur
A-
B-
C-
B. Predict the outcome if sequential feedback inhibition is occurring, where X inhibits
enzyme B and intermediate 1 inhibits enzyme A.
A-
B-
C-
Topic – Allosteric Proteins
To isolate a regulatory mutant of isopropyl malate synthetase (IPMS) enzyme it would be
best to plate 108 cells on medium containing
a.
b.
c.
d.
e.
leucine
fluoroleucine
methyl tryptophan
isoleucine
ketoisovalerate (KIV)
(10 points) Explain the “inhibition plus activation” mode of feedback inhibition
using Carbamyl Phosphate Synthetase as example. Make a diagram to represent CPS
as enzyme 2 (Figure 6.1 on page 158) using CTP, carbamyl aspartate, carbamyl
phosphate, glutamine, glutamate, ornithine, citrulline, and arginine as intermediates. Use
the letters in Fig. 6.1 to indicate the intermediates listed. Do not include other
intermediates
(20 points) You have been studying lilly kinase, the first enzyme in the gathman
triphosphate (gTP) synthesis pathway shown here:
lillic acid  lilly-phosphate     gathman triphosphate
1. Using gel permeation chromatography, the native form of the enzyme has been
isolated, and has an apparent MW of 140,000 D. The enzyme shows sigmoidal kinetics,
and is inhibited by gTP as shown in Figure 1A. When the protein is subjected to SDS
PAGE under denaturing conditions with -mercaptoethanol, two bands are seen; one
50,000 D and one 20,000 D.
2. In the presence of a small amount of urea, the kinetics remain sigmoidal (Figure 1B),
but no inhibition by gTP is observed. When sucrose gradient density centrifugation is
carried out in the presence of urea, two protein peaks are observed, and the activity is
associated with the one at 100,000 D (Figure 2).
3. If the native form is treated with DTNB, an agent that reduces (disrupts) disulfide
bridges, the enzyme preparation shows Michaelis-Menten kinetics with or without urea,
but is inhibited in the absence of urea (Figures 1C and 1D).
Give the sub unit structure of the enzyme. Draw the subunits as circles proportional to
their size. Indicate which subunit(s) have the active and the allosteric sites. Indicate the
possible nature of the subunit interaction. How is the quaternary structure maintained
(how are the subunits held together)? For each of the four enzyme activity plots, account
for the shape of the curve based on the subunit structure and cooperativity in binding.
Also explain any inhibition or lack thereof due to subunit structure.
Topic – Aspartate Family Regulation
Chymotrypsin treatment of aspartate kinase/ homoserine dehydrogenase I yields a protein
fragment that is
a.
b.
c.
d.
e.
AK+, HSD+, threonine sensitive
AK+, HSD-, threonine sensitive
AK+, HSD+, threonine insensitive
AK-, HSD-, threonine insensitive
AK-, HSD+, threonine insensitive
The product of the thrA gene aspartate kinase/ homoserine dehydrogenase I is
a. a multifunctional enzyme
b. an isoenzyme
c. inhibited by lysine
d. a and c
e. a and b
Aspartate Transcarbamoylase
Write the reaction carried out by this enzyme. What is the allosteric inhibitor?
Describe the subunit structure of the enzyme. Indicate size, number, and pertinent
binding activities of each subunit.
How are the subunits held together? How were they disrupted in the experiments?
Describe the experiment used to identify the regulatory subunits.
(10 points) Explain the regulation of isofunctional enzymes using aspartate kinases I and
III as examples. Make a diagram using aspartate, aspartyl phosphate, threonine,
homoserine, and lysine as intermediates A through E (not in that particular order) in
Figure 6.2 on page 309. Do not include other intermediates or AK II or homoserine
dehydrogenases.
Topic - Transcriptional Registration
Regulation of the maltose operon is an example of
a.
b.
c.
d.
activation of an operon by a positive regulator
attenuation
repression of an operon by a negative regulator
induction of an operon by an inducer
Which of the following is a gratuitous inducer (induces, but is not consumed) of the lac
operon?
a.
b.
c.
d.
X-gal
ortho-nitrophenyl galactoside
allolactose
isopropyl thiogalactoside
The leader peptide is synthesized when
a.
b.
c.
d.
anthranillate synthase is inhibited tryptophan
transcription is blocked by the tryptophan-corepressor complex
the antiterminator stem-loop 2:3 forms
the terminator stem-loop 3:4 forms
Operons that are controlled by termination/antitermination usually code for enzymes
involved in
a.
b.
c.
d.
catabolism of PTS sugars
catabolism of non-PTS sugars
synthesis of amino acids
none of the above
Matching mutants. Indicate the mutant on the bottom of the page which would exhibit
the phenotype given.
10. __________________ Uninducible by lactose, unable to rearrange lactose to
allolactose
11. __________________ A mutant is recovered from minimal media containing methyltryptophan. It shows low levels of the protein anthranillate synthase, and the enzyme is
not inhibited by tryptophan.
12. __________________ A constitutive mutant for the lac operon
13. __________________ A cryptic mutant, uninducible by lactose, but inducible by
IPTG and toluene.
14. __________________ A mutant is recovered from minimal media containing methyltryptophan. It shows high levels of the protein anthranillate synthase, and the enzyme is
inhibited by tryptophan.
15. __________________ A mutant that does not express the lac operon (and many other
auxiliary catabolic pathways). Expression does occur when cAMP is added.
16. __________________ This mutant produces a lac repressor protein that does not bind
the operator, thus it does not repress the lac operon.
17. __________________ A mutant that does not express the lac operon (and many other
auxillary catabolic pathways). Expression does not occur, even when cAMP is added.
crpcyalacIs
lacIlacOc
lacY
lacZ
trpA (active-site)
trpE (active-site)
trpE (allosteric-site)
trpR (allosteric-site)
(16 points) For each of the following mutant genotypes, describe the pertinent
phenotype. Estimate the level of β-galactosidase and acetylase produced as in the
homework problems. Include a diagram and a short explanation. Remember to include:
what the proteins are like; if the individual will be inducible, constituitive, or uninducible,
etc.; and how the level of activity would compare to wild type
A. cya+ crp+ lacI + o+ Z- Y+ A+ grown on glycerol and IPTG
B. lacI – o+ Z - Y+ A+ / F’ lacI + o+ Z+ Y+ A - grown on glycerol with and without IPTG
C. A cya mutant that does not bind enzyme IIIglc – P. How would it behave if grown on
lactose and glucose. Would the lac operon be expressed? Would lactose be transported?
D. A cya mutant grown on lactose and glucose. How would this affect the lag during
diauxic growth?
10 points
Discuss three levels of control of the synthesis of tryptophan that involve the amount and
activity of anthranillate synthetase in E. coli. Give an abbreviated pathway for
tryptophan biosynthesis, including erythrose-P, PEP, chorismate, anthranillate, and
tryptophan, and the enzyme. Give a gene map indicating the position of the operon and
the relevant control elements (genes).