Answers to challenge questions for Microbial Diversity, taught in spring 2018 by Prof Kristen DeAngelis at the University of Massachusetts Amherst

1. Key for activity reviews in
class
Microbiology 480
Microbial Diversity
Spring 2018

2. KEY: Activity for Review of
Unit 01.1 Founders of Microbiology
1. Visualizing microbes
a. Antonie van Leeouwenhoek
b. As a lens grinder, made the first microscope thus the first observation of
microbes
2. Sterile technique
a. Louis Pasteur
b. Using the swan-necked flasks, developed “pasterurization” as a way to sterilize
media, the first controlled cultivation
3. Culturing single colonies
a. Robert Koch
b. With his assistant Julius Richard Petri, developed agar plates for isolating single
colonies, or cultures derived from single cells, the first isolation of microbes
4. Method for defining causative agents
a. Robert Koch
b. Developed “Koch’s postulates” as a way of showing causation of disease-causing
mirobes, but process is often applied to other scenarios
5. Sequencing methods for true phylogenies
a. Carl Woese
b. Developed primers for PCR Sanger sequencing of the small subunit ribosomal
RNA gene, which revealed the very divergent new (at the time) domain Archaea

3. KEY: Activity for Review of
Unit 01.2 Measures of Diversity
Draw a picture of the Woesian Tree of Life, and
annotate it with labels including
a. The contribution of Carl Woese – 3 domains of life
b. The contribution of Ernst Haeckl – archaea +
bacteria = protista or prokaryotes
c. The contribution of Carl Linnaeus - DKPCOFGS
Bacteria
Archaea
Eukaryotes

4. KEY: Activity for Review of
Unit 01.3 Extent of Diversity
For each set of pairs, which environment has the
most microbes? Which has the most diversity? Why?
1. Salt marsh likely has more micorbes than the open ocean because there
is more food (from plant root exudates and decomposing plant litter). For
this reason, there is likely to be more diversity in the salt marsh, too, because
there is more diversity in niches: food source, aerobic and anaerobic
environments, surface and free-living spaces.
2. Top, surface soil will have more microbes than deep soil, because there is
more food (from soil organic matter, plant root exudates and plant litter).
Deep soil might have similar diversity because both environments have a
range of niches, but deep soil has more minerals and more anaerobic sites.
3. The gut has almost all of the microbes in the human microbiome, so this
site will certainly have more microbes compared to skin. But because of
the diversity of niches in skin, the diversity of the microbes there might be
comparable. It likely depends on the host’s diet health.

5. KEY: Activity for Review of
Unit 02.1 Reading Trees
1. Circle the groups included in the Prokaryotes.
2. The group Prokaryote is
a. Monophyletic – all have a common ancestor.
b. Polyphyletic – species have many common ancestors.
c. Paraphyletic - group excludes one ancestor.
The
term
prokaryote
defines
organisms
by
what
do
not
have
(nuclei).
It
is
be=er
not
to
measure
organisms
by
their
form
or
func?on,
because
these
traits
can
be
plas?c
(changing).
Nuclei
are
not
an
ancestral
trait.

6. KEY: Activity for Review of
Unit 02.2 Making Trees
1. Which tree is a more likely
representation of
Methanopyrus kandleri?
Why?
The top tree, because the branche
for M. kandleri have higher
bootstrap values
2. What could explain the
differences between the
two trees?
Perhaps one of the genes was
horizontally transferred, meaning
that it is not a true phylogeny.
One gene could be under selective
pressure or could have a different
function in one set of organisms
compared to the others.

7. KEY: Activity for Review of
Unit 02.3 Rooting Trees
• What can we infer about the biology of the Last
Universal Common Ancestor based on the fact that
different genes place the root in different Domains?
– LUCA had characteristics of both Bacteria and Archaea
– Because DNA sequence-related genes puts the root
within the Bacteria, the LUCA likely had DNA-based
functions (replication, for example, elongation factors)
more like modern day Bacteria
– LUCA will share other characteristics with bacteria that
root the tree this way, e.g., ATPases
– Because protein sequence puts the root of the tree of
life within the Archaea, the LUCA likely had proteins
that looked more like Archaea
– Same is true for tRNA, 5S, Rnase P: LUCA would have
looked more like Archaea

8. KEY: Activity for Review of
Unit 03.1 Early Earth
• Place the following events in the history of Earth in order:
__3__ First eukaryotes with endosymbiotic Rickettsiales (first
mitochondria)
__2__ First plants with endosymbiotic cyanobacteria (first chloroplasts)
__1__ First eukaryotes
__4__ First land plants
• First plants would have to come before first land plants
• Plants are eukaryotes with chloroplasts, so first eukaryotes would
have to come before plants or land plants
• 2.5 Ga – First eukaryotes (cells with nucleus)
• 1.5 Ga – First eukaryotes with endosymbiotic Rickettsiales (Sar11
clade) bacteria, now mitochondria: symbiogenesis
• 1.5-1 Ga – First plants with endosymbiotic cyanobacteria
(chloroplasts)
• ~475 Ma – first land plants

9. KEY: Activity for Review of
Unit 03.2 Evolution of Cellular Life
• What were some characteristics of protocells? In answering,
describe if it had:
– DNA – no, DNA was the last biomolecule to evolve
– RNA – yes, would have had RNA acting catalytically as well as
encoding some functions
– Lipids – almost certainly; lipid sacs called micelles form
naturally
– Membranes – we assume there was a membrane bilayer with
proteins in the membrane
– Proteins – no, because the presence of proteins is the mark of
a cell, used for metabolism and import into the cell
– Replication – yes, even early protocells “replicated” as they
travelled currents from warm to cool
• Was LUCA a protocell? Why or why not? – the last
protocell would have been separated from LUCA by up to a
billion years (4Ga for first life, 3Ga for first “prokaryote”, or
LUCA).

10. KEY: Activity for Review of
Unit 03.3 Evidence for Early Life
How do we know that the first life on Earth occurred about
3.5 Ga?
• Old rock deposits are identified by radiogenic dating. Once
they are identified as very Old Rock (over 3 Ga), isotopic
fractionation can be used to show that the carbon in the
rock is organic.
• Microfossils may be found in old rock deposits identified by
radiogenic dating. Once they are identified as very Old Rock
(over 3 Ga), isotopic fractionation shows that the
microfossils are organic and thus microbial cells.
• Some lipids or other complex molecules are only found in
microbes. If these are found in Old Rock, this can be used
to date early life.
• Phylogenetic trees can act as molecular clocks. A good tree
can be used to root the tree of life and estimate the age
of early life, based on assumptions about the rate of
mutation (a proxy for the rate of evolution).

11. KEY: Activity for Review of
Unit 04.1 Biofilms
Draw a picture of how a biofilm “cycles” as a form of
dispersing. Include in your diagram labels for the five
stages of biofilm growth.
1. Initial attachment,
2. Irreversible attachment,
3. Maturation,
4. Recruitment, and
5. Dispersion.
1
2
3
4
5

12. KEY: Activity for Review of
Unit 04.2 Motility
We discussed four types of flagellar arrangements earlier. What
type do you think spirochaetes have, and why?
• The four types are
A. Monotrichous bacteria have a single flagellum (e.g., Vibrio
cholerae).
B. Lophotrichous bacteria have multiple flagella located at the
same spot on the bacteria's surfaces which act in concert to
drive the bacteria in a single direction.
C. Amphitrichous bacteria have a single flagellum on each of two
opposite ends (only one flagellum operates at a time, allowing
the bacteria to reverse course rapidly by switching which
flagellum is active).
D. Peritrichous bacteria have flagella projecting in all directions
(e.g., E. coli).
Spirochaetes can be monotrichous, lophotrichous or amphitrichous.
The important characteristics are that (1) the flagella does not
emerge from the outer membrane, and (2) is located only at one or
both ends

13. KEY: Activity for Review of
Unit 04.3 Managing Movement
• Which statements are true about quorum
sensing? (Circle all that apply.)
a. Quorum sensing enables bacteria to co-
ordinate their behavior. YES.
b. The QS signal is constitutively produced at
a low levels. YES.
c. Quorum sensing genes always occur in
pairs of synthase and receptor genes. YES.
d. Different QS signals may elicit different
behaviors. YES.

14. KEY: Activity for Review of
Unit 05.1 Geographic distance
This is an aerial photo of the Don Juan Pond in
Antarctica has 40% salinity; this is the saltiest known
body of water on Earth.
1. This is a C-limiting environment. What kind of
compatable solutes do microbes make here?
– A microbe experiencing osmotic stress in conditions
where carbon is limiting will likely make compatible
solutes that are non-carbon based. This should include
Phosphorous (K+) and Sodium (Na+).
2. Would you expect this environment to have high or
low diversity? Why?
– This environment should be low in diversity, because
salinity is the strongest driver of microbial community
structure, and special funcitons are required for living
in high salinity environments.

15. KEY: Activity for Review of
Unit 05.2 Community distance
Some marine bacteria display bipolar distributions in the
Earth's oceans, occurring exclusively at the north and south
poles and nowhere else. Is this evidence for or against the
Baas-Becking Hypothesis? Explain, and be sure to restate the
Baas-Becking Hypothesis.
• This observation is evidence for the Baas-Becking
Hypothesis, which is that “everything is everywhere but the
environment selects.”
• If everything were everywhere, then it all types of
organisms are present at even very small abundances in all
environments. If the environment selects, then this bipolar
distribution (evident only at the two poles) might suggest
that there is something special about the poles that
encourages the growth of the marine bacteria.

16. KEY: Activity for Review of
Unit 05.3 Linking function to phylogeny
If you wanted a measure of microbial functions in a natural
environment, which method of sequencing would be
appropriate? Circle all that apply.
• Comparative genomics – NO, this measure of sequencing genomes of
isolated organisms cannot now be done in mixed, natural communities
• Metagenomics – YES, sequencing all the DNA from a mixed community
will give you an estimate of possible functions
• Metatranscriptomics – YES, also called RNAseq, this sequencing of all
RNA is a measure of genes transcribed
• Phylogenetics – NO, this measure of gene markers of evolution only
gives information on function by proxy and is not a direct measure of
microbial function
• Stable isotope probing – YES, perhaps the best measure of microbial
function, if the substrate for the funciton of interest can be added as
an isotopically labeled substrate

17. KEY: Activity for Review of
Unit 06.1
• Draw a photosynthetic microbial mat, and map out the layers to
show how the primary producers at the top indirectly provide
energy and carbon to the organisms at the bottom.
Top green layers = green algae, cyanobacteria
- fix C, exude sugars that may be available to lower layers
Pink layer = purple sulfur bacteria
- anoxygenic phototrophs, oxidize H2S to S or SO4 for energy
Orange layer = spirochaetes
- microaerophilic heterotrophs
Purple layer = green sulfur bacteria
- photosynthetic, oxidize sulfide for reducing equivlaents
Black layer = sulfate reducers
- reduce SO4 to H2S obtained from upper layers
Bottom is iron sulfide-rich waste products and decaying mats

18. KEY: Activity for Review of
Unit 06.2
1. Chloroplasts are derived from an ancient
endosymbiotic event, and belong to the
Phylum Cyanobacteria (blue-green algae).
2. Like the cyanobacteria, they fix carbon via
the calvin cycle, and make reducing
equivalents and energy (ATP) from light using
oxygenic photosynthesis, which employs the
two photosystem, Z-scheme of electron
transport from the reaction center, to
chlorophyll a to the FeS center to the
electron transport chain.

19. KEY: Activity for Review of
Unit 06.3
• The Aquificae and Thermotogae are both primitive and deep-
branching phyla. Can you draw a tree with deep branches that
are not primitive, and with primitive branches that are not deep?
Do you know of any examples of either?
• A great example of deep, long branches is the eukaryotic tree, in
which the deep branches are long (and mostly parasitic) and the
plants and animals are relatively short branches. An open question
in evolution is: what exactly IS a “deep” branch? If you add a
large collection of sequences to the Thermotoga lineage (say, all
known species in this group) and prune many of the sequences
from the rest of the tree, only the Aquificae branches look “deep”
now. Primitive species have short branch lengths, so are more
closely related to the common ancestor. Deep branches have
direct lineages closer to the root of the Tree of Life. An organism
can be deeply rooted, or primitive, or both.

20. KEY: Activity for Review of
Unit 07.1
• How are soil and sediment related?
– Sediment is soil moved by water.
• What is the name for soil influenced by
plant roots? Rhizosphere soil.
• Why are the acid-rich sediments of the Rio
Tinto considered analog sites for studying
possible life on Mars?
– Due to a mining accident, the Rio Tinto is replete
with heavy metals and iron. Anoxic conditions in
the sediment replicate anoxic environments on
Mars. Because there seems to have been water
on Mars in the past, the Rio Tinto could look like
Martian sediment from past eons.

21. KEY: Activity for Review of
Unit 07.2
• What microbe is an environmental biocontrol
agent for mosquito-transmitted viral
pathogens? Wolbachia, an Alphaproteobacteria
• What microbe is the ancestor of the
mitochondria? Rickettsia, an
Alphaproteobacteria
• What microbe is responsible for ulcers?
Helicobacter pylori, an Epsilonproteobacteria
• What phylum and class do the purple sulfur
bacteria belong to? The Gammaproteobacteria,
for example Chromatium spp.

22. KEY: Activity for Review of
Unit 07.3
• For each microbe listed, name the phylum
it belongs to, and match it to its
function.
_ Bacillus cereus - Soil microbe that produces
endospores
_ Mycobacterium tuberculosis - Animal
pathogen with no known environmental
reservoir
_ Streptomyces antibioticus - Most common
source of industrial antibiotics

23. KEY: Activity for Review of
Unit 08.1
Match the rare phylum-associated bacteria with its relatively
unique morphology. Taxa may be used more than once.
1. Elemental bodies - B
2. Extreme tolerance to UV irradiation - D
3. Pirrelulosomes – A
4. Riboplasm - A
5. Taq polymerase - C
6. Division by septal curtain - D
a) Blastopirellula marina
b) Chlamydia
c) Thermus aquaticus
d) Deinoccus radiodurans

24. KEY: Activity for Review of
Unit 08.2
Which are true of the eocyte hypothesis?
a. It presents a competing view of the origin of
the domain Eukarya. TRUE
b. It presents a competing view of the structure
of the domain Eukarya. FALSE: the eocyte tree
still has 5 eukaryotic supergroups.
c. It suggests that the closest relatives to the
Eukarya are the eocytes. TRUE.
d. It is a different understanding of the root of
the tree of life and thus LUCA. FALSE: the root
is still in the bacteria, determined by
constructing trees from ancient paralogous
proteins.

25. KEY: Activity for Review of
Unit 08.3
1. What are some reasons why bacteria from the rare phyla
remain uncultivated?
• As with the Chlamydia, uncultured microbes might have undergone
reductive evolution and rely on a host to reproduce
• As with Thermus, high temperatures and oligotrophic (slow)
growth might make cultivation without contamination difficult
• Cryptic micronutrient requirements, Auxotrophy, or require specific
metabolic partners
• As with Planctomycetes and Deinococcus, it may not be clear why
they remain uncultivated.
2. For each pair of phyla below, circle the phylum that is the
most phylogenetically diverse.
• Proteobacteria or Chlorflexi
• Deinococcus or Firmicutes
• Chlorobi or Bacteroidetes
• Candidate Phylum Radiation or Chlamydia

26. KEY: Activity for Review of
Unit 09.1
• How do we get our microbiomes?
– Babies are born with very few microbes, and
acquire their microbiome from their mothers at
birth. Microbes are constantly introduced from the
outside, but our microbiome helps our human cells
to acts as a barrier.
• How does the human microbiome functional
diversity compare to the phylogenetic diversity.
– Despite variation in community structure
(phylogenetic diversity), metabolic pathways tend to
be stable among individuals (functional diversity).

27. KEY: Activity for Review of
Unit 09.2
• Match the eukaryotic superkingdom to its major
characteristics
1. Excavates - b
2. Chromalveolates
- a
3. Plantae - e
4. Rhizaria - c
5. Unikonts - d
a) mostly phototrophic algae,
diatoms
b) flagellated single-celled
eukaryotes, pathogens
c) all unicellular eukaryotes,
very diverse
d) include Amoebozoa and
Opisthokonts, which have two
main groups, fungi animals
e) plants, including land plants,
green and red algae; all have
plastids (chloroplasts) derived
from cyannobacteria

28. KEY: Activity for Review of
Unit 09.3
• Compare the mechanism of action for
probiotics and prebiotics. Which is proven to
be effective in changing the microbiome?
– Probiotics are live microbes that are eaten, and
prebiotics are foods that the host cannot eat,
but feed specific microbes in the gut
microbiome.
– Although some probiotics have shown promise in
research studies, strong scientific evidence to
support specific uses of probiotics for most
health conditions is lacking.

29. KEY: Activity for Review of
Unit 10.1
• Which statements are true about
permafrost?
a) It has an active layer which freezes and
thaws in cycles. - TRUE
b) It is colonized by bacteria, archaea and
eukarya. - TRUE
c) Soils frozen for two or more weeks are
permafrost. – FALSE, two or more YEARS
d) Permafrost can be frozen soils but not frozen
sediments. – FALSE they can be either or a
mix

30. KEY: Activity for Review of
Unit 10.2
1. Name one phylum or class of Archaea.
– Crenarchaeota, Euryarchaeota, Thaumarchaeota,
Koryarchaeota, and Nanoarchaeum
2. For the list of Archaeal traits below, circle
which ones are shared with the Eukarya
a) Information-processing machinery
b) Ether-linked membrane lipids (this trait is unique
to Archaea)
c) Cytoskeleton (this trait is shared with Bacteria, not
Eukarya)
d) Nucleosomes
e) Flagellar structure (this trait is unique to Archaea)

31. KEY: Activity for Review of
Unit 10.3
What are the effects of thawing permafrost?
• Permafrost stores twice as much C as what is in
the earth’s atmosphere right now. The biggest
effect of thawing permafrost is to the carbon
cycle. Thawing permafrost will release
geothermal CH4, a potent greenhouse gas.
Though in the short term, plant growth on
thawed soils will be a sink for C, thawing
permafrost will also increase microbial activity,
releasing CH4 and CO2. Thawing permafrost will
also release mercury and cause the growth of
microbes frozen or dormant for thousands of
years. This includes spore-forming bacteria and
viruses.

32. KEY: Activity for Review of
Unit 11.1
• Match the virus with the hypothesis of origin
of evolution that best describes it.
1. __c__ Mimivirus
2. __b__ HIV
3. __b__ Bacteriophage Mu
4. __a__ Rhinovirus
5. __c__ Klosneuvirus
6. __a__ Rice yellow mottle virus-associated viroid
a. Virus-first hypothesis
b. Progressive hypothesis
c. Regressive hypothesis

33. KEY: Activity for Review of
Unit 11.2
Describe how Koch’s postulates were attempted to be applied to the
discovery of prions as the infectious agent in Mad Cow disease
(Bovine Transmissible spongiform encephalopahty, or TSE). How might
one come closer to satisfying Koch’s postulates?
• Koch’s postulates are the way to prove causation, in this case the
causative agent of infection. To satisfy Koch’s postulates, you’d
have to isolate the infectious agent from an organism with the
disease, re-infect an organism and recreate the disease state,
then re-isolate the same infectious agent.
• In this case, the infectious agent is an alternate conformation of
a folded protein. It is not so easy to measure the folding of a
protein; usually this requires x-ray crystallography.
• This is also complicated by the fact that TSE can arise
spontaneously.

34. KEY: Activity for Review of
Unit 11.3
• What is the difference between a virus and a viroid?
– Viruses are larger
– Viruses can be DNA or RNA, viroids are ssRNA
– Unlike viruses, viroids have no capsid or protein shell
– Viruses infect organisms from three domains, viroids infect
only plants (that we know)
• What is the difference between a viroid and a
satellite?
– Viroids direct the host cell to help with replication, satellites
depend on a helper virus
– Satellites can be DNA or RNA, viroids are ssRNA
– Unlike satellites, viroids have no capsid or protein shell