Unit 8: Rare and Uncultured Microbes
LECTURE LEARNING GOALS
1. Describe the phyla containing rare bacteria: Deinococcus/Thermus, Chlamydia & Planctomycetes.
2. Describe the sequencing methods used to understand uncultured microbes. Explain the Eocyte hypothesis and how this model differs from the three domain tree of life.
3. For the cultured microbes, describe major characteristics for the 13 bacterial phyla, and explain why some microbe remain uncultivated.
6
1. Be a Microbio
Peer Advisor!
• Work with a great team.
• Practice leadership skills.
• Use your unique skills to build community.
• Help us all stay connected.
• Show your enthusiasm for microbiology.
Applications due March 8th!
http://bit.ly/micropa_app
Or contact Heather Reed at
hreed@umass.edu
2. COVID Conversations:
A Q&A on the Microbiology
of COVID-19
Tuesday, March 16th
@ 5 pm
Hosted by Dr. Mandy Muller and
Dr. Wilmore Webley of the Microbiology Department
Register using barcode below or
at www.micro.umass.edu !
3.
4. The SARS-CoV-2 genome is highly reduced…
and has no 16S ribosomal RNA genes
ul Qamar et al., 2020
5. RARE AND UNCULTURED MICROBES
Unit 08, 3.2.2021
Reading for today: Brown Ch. 13 & 14
Reading for next class: Brown Ch. 16, Walter & Ley (moodle)
Dr. Kristen DeAngelis
Office Hours by appointment
deangelis@microbio.umass.edu
Poll Q
6. Unit 8: Rare and Uncultured Microbes
LECTURE LEARNING GOALS
1. Describe the phyla containing rare bacteria:
Deinococcus/Thermus, Chlamydia &
Planctomycetes.
2. Describe the sequencing methods used to
understand uncultured microbes. Explain the
Eocyte hypothesis and how this model differs
from the three domain tree of life.
3. For the cultured microbes, describe major
characteristics for the 13 bacterial phyla, and
explain why some microbe remain
uncultivated.
6
7. Unit 8: Rare and Uncultured Microbes
LECTURE LEARNING GOALS
1. Describe the phyla containing rare bacteria:
Deinococcus/Thermus, Chlamydia &
Planctomycetes.
2. Describe the sequencing methods used to
understand uncultured microbes. Explain the
Eocyte hypothesis and how this model differs
from the three domain tree of life.
3. For the cultured microbes, describe major
characteristics for the 13 bacterial phyla, and
explain why some microbe remain
uncultivated.
7
9. Deinococci, Chlamydia &
Planctomycetes
• There are 13 main phyla, and we are
talking about three with few cultured
representatives
• The Chlamydia & Planctomycetes are
closely related
• Deinococcus-thermus is one of the
more deeply branching phyla.
11. Phylum Deinococcus-Thermus
• There are two well-known genera in this
phylum, Deinococcus and Thermus
– These two genera are phenotypically and
phylogenetically quite different
• Examples
– Deinococcus radiodurans
– Thermus aquaticus
13. Deinococcus radiodurans
• D. radiodurans survives high exposure to
gamma-irradiation
– E. coli can withstand up to 500 Gy (Grays,
Joules per kilogram)
– For comparison, 10 Gy is lethal to humans
– maintains several copies of the each of its two
chromosomes, as the mechanism of DNA
stress resistance
• cells divide by forming a septal curtain
– It closes in like the shutter of a camera
– There are two perpendicular curtains formedin
cell division, producing tetrads
14. Thermus aquaticus
Phylum Deinococcus-Thermus
• Isolated from many
alkaline hot springs in
Yellowstone NP
• Pink colonies, especially
when grown in light due to
pigments
• Its DNA polymerase is
highly heat resistant and
error-correcting
– Taq polymerase is
commonly used in PCR
16. Phylum Planctomycetes
• Diversity is unclear, because they are so few
cultivated
• Metabolism of almost all are aerobic,
heterotrophic, mesophilic oligotrophs
• Habitats are mostly aquatic and especially
eutrophic environments, though sequences
are detected in a wide range of
environments including wastewater and soils
17. Blastopirellula marina
Phylum Planctomycetes
• Common freshwater species
• Reproduce by budding
• Internal membrane-defined
compartmentalization
– Central pirellulosome contains
the riboplasm and nucleoid
(genome)
– Riboplasm contains ribosomes
and DNA
– Paryphoplasm contains RNA but
not ribosomes
19. Phylum Chlamydiae
• Low phylogenetic and phenotypic diversity
– Few cultured representatives
– Uncultured diversity seems to be much greater
• Metabolism
– Greatly reduced genomes
– Remain capable of
• information processing (transcription, translation,
replication)
• cell envelope
• central metabolism
• Habitat: Obligate intracellular parasites
transmitted via small, metabolically inert
particles
20. Chlamydia trachomatis
Phylum Chlamydiae
• Human pathogen that
causes the most common
sexually transmitted disease
(STD) in the U.S.
• Most infections are
asymptomatic, but
untreated infections can
cause sterility
• Repeated ocular infection
in children can cause
blindness
C. trachomatis elementary bodies attached to human sperm.
From Courtney S. Hossenzadeh in Microbiology Today.
22. Developmental cycle of Chlamydia
• Biphasic life cycle
– Elemental bodies are infectious and metabolically inert
– Replication bodies are much larger, noninfectious and
osmotically fragile
• RBs live inside the cell, metabolize, grow and and
divide within the endocytic vessicles
• When resources in the cell become limited, most
RBs differentiate into EBs and are released from the
cell
• Not all infectious cycles end in host cell lysis; some
species are released by exocytosis
23. Reductive evolution
• Over evolutionary time,
parasites rely more on
the host for the things it
needs and may simplify
its genome
• This reduction allows the
organism to devote
more resources to
reproduction
24. Activity for Review of
Unit 08.1 Rare phyla
Ernst Haeckl’s Tree of Life
was wrong because it’s based
on morphology. Today,
scientists are using Machine
Learning to identify microbes
by morphology. What are
some distinct morphologies,
and which organisms are they
linked to?
24
25. Unit 8: Rare and Uncultured Microbes
LECTURE LEARNING GOALS
1. Describe the phyla containing rare bacteria:
Deinococcus/Thermus, Chlamydia &
Planctomycetes.
2. Describe the sequencing methods used to
understand uncultured microbes. Explain the
Eocyte hypothesis and how this model differs
from the three domain tree of life.
3. For the cultured microbes, describe major
characteristics for the 13 bacterial phyla, and
explain why some microbe remain
uncultivated.
25
Poll Q
27. Bacterial phyla with few to no
cultivated representatives
• The 13 “main” phyla were described by Carl Woese
in his classic 1987 paper
• Since then, sequencing revealed that microbial
diversity is much greater than this!
• Most cultivated, characterized bacteria fall into one
of five phyla
– Proteoabcteria, Firmicutes, Actinobacteria, Bacteroidetes
and Cyanobacteria
– Animal diversity is similar: most animal species belong to a
few animal phyla e.g. nematodes (round worms) and
arthropods (insects)
28. Molecular approaches aka ‘Omics
• Amplicon-based sequencing
– limited capacity for discovery
– Better for phylogenetics because traits can be aligned
• Shotgun sequencing
– Lots of room for discovery
– Half of sequences have no known homology
29. Assembling whole genomes from
metagenomic data using binning
• Short sequences are
“binned” based on
shared characteristics
– GC content
– taxonomy
– Coverage (sequence
depth)
– K-mer frequency
http://dx.doi.org/10.3389/fmicb.2015.01451
30. Detecting unculturable bacteria
• Genomics – sequencing whole genomes, DNA
• Transcriptomics – sequencing RNA from genomes
• Proteomics – sequencing proteins
• Metabolomics – identification of all metabolites,
usually by analytical chemistry
• Meta-
– Add the prefix “meta-” to any of the above, and it refers to
sequencing mixed communities instead of single species
– For example, Metagenomics – sequencing mixed
communities
– For example, Metatranscriptomics – sequencing RNA from
mixed communities
33. A “new” tree of life
• Shotgun sequencing from a diversity of
environments, including filtration to include
very small organisms
• Based largely on metagenome-assembled
genomes (MAGs)
• Tree constructed based on aligned and
concatenated a set of 16 ribosomal protein
sequences from each organism
• Two surprising attributes
– Topology: Recovery of a two-domain tree of life
– Uncultured diversity is the majority (e.g. the CPR)
34. 3 Domain ToLs
• Woesian tree (top)
– Based on structural RNA
sequences
• Alternative 3-domain ToL
(middle)
– Based on protein structures
• NOT a ToL (bottom)
– No tree places the root in
the Eukarya
– “Prokaryote” is NOT a
monophyletic group
– LUCA was not a eukaryote
34
34
Bacteria Archaea Eukaryotes
root
Bacteria
Archaea Eukaryotes
root
Bacteria
Archaea
root
36. Eocyte hypothesis
• some analyses of the protein translation elongation
factors, paralogs used to root the 3-domains tree,
do not actually recover the 3 domains
• These show a tree where the eukaryotic proteins
branch as phylum of Archaea called the
Crenarchaeota (aka the eocytes)
• shapes of ribosomes in the Crenarchaeota and
eukaryotes are more similar to each other than to
either bacteria or the second major kingdom of
archaea, the Euryarchaeota.
37. The two-domain Eocyte ToL
vs the three-domain ToL
37
Eukarya Eukarya
Bacteria Bacteria
Archaea Archaea
38. • These are competing hypotheses for the origin of
the domain Eukarya.
– (A) The rooted 3-domains tree posits that the Archaea,
consisting of 2 kingdoms Euryarchaeota and
Crenarchaeota (eocytes), are monophyletic and more
closely related to the eukaryotes than to Bacteria.
– (B) An alternative hypothesis, the eocyte tree, posits that
the Archaea are paraphyletic, with the eocytes
(Crenarchaeota) most closely related to the eukaryotes.
– Paraphyletic = monophyletic except for one group
Cox, C. J., Foster, P. G., Hirt, R. P., Harris, S. R., Embley, T. M. (2008). "The archaebacterial
origin of eukaryotes". Proc Natl Acad Sci U S A 105 (51): 20356-61.
The two-domain Eocyte ToL
vs the three-domain ToL
40. The Eocytes
• Domain Eukarya still has 5 supergroups: Excavates,
Chromalveolates, Plantae, Rhizaria, & Unikonts
– 92 bacterial phyla – we cover 13 in this class !
– 25 Archaeal phyla
• Crenarchaeota (aka the Eocytes)
• In this tree, Archaea are a polyphyletic group
– Euryarchaeota
– Other phyla including Thaumarchaeota, Nanoarchaeum,
Koryarchaeum
40
41. Activity for Review of
Unit 08.2 Eocyte hypothesis
• Based on what you know about how to make a
tree, and how to make and root a tree of life,
why do you think that there are so many
models (trees) of the phylogeny of life?
41
42. Unit 8: Rare and Uncultured Microbes
LECTURE LEARNING GOALS
1. Describe the phyla containing rare bacteria:
Deinococcus/Thermus, Chlamydia &
Planctomycetes.
2. Describe the sequencing methods used to
understand uncultured microbes. Explain the
Eocyte hypothesis and how this model differs
from the three domain tree of life.
3. For the cultured microbes, describe major
characteristics for the 13 bacterial phyla, and
explain why some microbe remain
uncultivated.
42
Poll Q
44. Hug et al. 2016. Nature Microbiology.
New View of the Domain Bacteria
• And a new phylum: the Candidate Phyla Radiation
– “CPR”
– all members have small genomes and most have restricted
metabolic capacities.
– Most are likely symbionts, with greatly reduced genomes.
• A striking feature of this tree is the large number of
major lineages without isolated representatives (🔴).
– Uncultivated organisms clearly comprise the majority of
life’s current diversity
• Domain Bacteria includes more major lineages of
organisms than the other Domains.
– Domain bacteria is the most diverse domain
48. Bacteroidetes and Spirochaetes
• Bacteroidetes
– Very diverse phylum
– mostly anaerobic fermenters
– Common in guts of animals
• Spirochaetes
– Found in sediments and some are pathogens
– Mostly heterotrophs with internal polar
flagella
– May be progenitors for eukaryotic flagella
50. Green phototrophic bacteria
• Chloroflexi (green non-sulfurs)
– thermophilic phototrophs and heterotrophs
– Single type of photosystem, Cyclic photophosphorylation to get
energy (ATP) from light
– Most fix C via the hydroxypropionate pathway
• Chlorobi (green sulfurs)
– Fix C using H2 or sulfur as electron donors for reverse TCA cycle,
strict photolithoautotrophs
– Energy is generated via cyclic photophosphorylation
– Most can fix N
• Cyanobacteria
– All carry out oxygenic photosynthesis with two photosystems to
get energy and reducing power
– fix CO2 via the Calvin cycle
– Most can fix N
54. Proteobacteria
• Phylum so diverse that they are mostly are discussed
by classes
– Alphaproteobacteria
– Betaproteobacteria
– Gammaproteobacteria
– Deltaproteobacteria
– Epsilonproteobacteria
• In the new ToL, the phylum proteobacteria is not
monophyletic!
– Because of this the classes are identified individually.
– For example, the Deltaproteobacteria branch away from
the other Proteos.
59. Activity for Review of
Unit 08.3 Rare bacteria
The Candidate Phyla Radiation (CPR) contain
the most deeply-rooted organisms in the
bacteria. What kind of traits do you predict
that they will have?
59
60. Unit 8: Rare and Uncultured Microbes
LECTURE LEARNING GOALS
1. Describe the phyla containing rare bacteria:
Deinococcus/Thermus, Chlamydia &
Planctomycetes.
2. Describe the sequencing methods used to
understand uncultured microbes. Explain the
Eocyte hypothesis and how this model differs from
the three domain tree of life.
3. For the cultured microbes, describe major
characteristics for the 13 bacterial phyla, and
explain why some microbe remain uncultivated.
Next class is Unit 9: Diversity of the Human Microbiome
Reading for next class: Brown Ch. 16, Walter & Ley (moodle) 60