This document provides an overview of a microbial genomics course project involving bacterial isolates from a long-term soil warming experiment. The project aims to sequence genomes of isolates collected from control and warmed soil plots over the course of the experiment to investigate evidence of microbial adaptation to warming at the genomic level. Isolates representing different time points in the experiment may allow insights into evolutionary responses to warming over time. The document outlines the workflow for building a culture collection from the soil samples and selecting isolates for genome sequencing based on phylogenetic and physiological analyses.

1. Meet the Isolates!
Introducing our capstone genomes
MICROBIO 590B Bioinformatics Lab: Bacterial Genomics
Professor Kristen DeAngelis
UMass Amherst
Fall 2022
1

2. Lecture Learning Goals
• Explain how the Earth’s climate is changing, and describe the
symptoms of climate change.
• Define soil, and explain why it is an important element in healthy
ecosystems as well as a possible climate solution.
• Describe the long-term warming field experiment ongoing at the
Harvard Forest in central Massachusetts.
• Explain the evidence for bacterial adaptation to long-term warming.
• Describe the origin of the genomes that you are going to work with
for your capstone projects, and describe how genomes were chosen
for genome sequencing.
2

3. The Earth’s climate is warming
• We are now in the Anthropocene, a proposed new epoch that
describes the influence of humans on earth system
• We know that increased fossil fuel burning is releasing too much CO2
into the atmosphere, causing a greenhouse effect
• The question is, will microbes act as a self-reinforcing feedback to the
climate system?
Climate.NASA.gov

4. The Earth’s climate is warming
• We are now in the Anthropocene, a proposed new epoch that
describes the influence of humans on earth system
• We know that increased fossil fuel burning is releasing too much CO2
into the atmosphere, causing a greenhouse effect
• https://climate.nasa.gov/climate_resources/240/the-greenhouse-effect-simplified/
• The question is, will microbes act as a self-reinforcing feedback to the
climate system?
Climate.NASA.gov

5. 5

6. The Greenhouse effect: Earth is Goldilocks
6
Not enough greenhouse effect
Mars has a very thin atmosphere, nearly all carbon dioxide.
Because of the low atmospheric pressure, and with little to no
methane or water vapor to reinforce the weak greenhouse effect,
Mars has a largely frozen surface that shows no evidence of life.
Too much greenhouse effect
The atmosphere of Venus, like Mars, is nearly all carbon dioxide. But
Venus has ~154,000 times as much CO2 in its atmosphere as Earth (and
~19,000 times as much as Mars does), producing a runaway
greenhouse effect and a surface temperature hot enough to melt lead.

7. Climate change is not the Apocalypse
• Temperatures will continue to rise
• Trends towards increased heavy
precipitation events, along with more
droughts and more heat waves
• Hurricanes will become stronger and
more intense
• Sea level will rise 1 to 8 feet by 2100
• Length of the frost-free season (and
growing season) will lengthen
• The Arctic will become ice-free
• Changes will continue, depending upon
how much we can curb fossil fuel
emissions
7
https://www.masslive.com/weather/2020/06/holy-flash-flooding-massachusetts-residents-share-photos-of-golfball-sized-hail-lightning-strikes-and-street-flooding-from-sunday-night-storm.html
https://climate.nasa.gov/effects/

8. Soils
• Store three times as much
C as the atmosphere
• Microbial activity regulates
soil C storage or loss
• sensitive to the
environment (ecology)
• may change over time
(evolution)
8
IUSS.org, SSSA.org

9. Soil is a Natural Climate Solution
Griscom et al, PNAS 2017
• NCS will help stabilize
warming to below 2oC, the
amount of warming required
for a stable climate & in the
Paris Climate Agreement
9

10. Will soils be a self-reinforcing feedback to climate?
• A lot of uncertainty in
forecasting soil future
response to climate
change is due to
microbial response
Freidlingstein et al, 2006

11. Harvard Forest Warming Experiments
36m2, 6 replicates
Barre Woods: 18
years
900m2, 1 replicate
Prospect Hill: 30 years
SWaN plots: 15 years
9m2, 6 replicates

14. Measuring soil respiration in response to
+5oC soil warming at Harvard Forest
• 70-80% respiration is microbial
• Melillo et al 2002, 2011
• Thermal adaptation of respiration
• Bradford et al., 2008
• Decreased labile soil organic
matter
• Frey et al 2008, Melillo et al 2011
• Decreased fungal biomass
• Frey et al 2008
Melillo et al., Science 2017
Δ(Htd
–
Ctl)
Soil
CO
2
flux
(g
CO
2
-C
m
-2
y
-1
)

15. Soil C loss has been discontinuous and non-linear over
30 years of warming
• 70-80% respiration is
microbial
• Melillo et al 2002, 2011
• Thermal adaptation of
respiration
• Bradford et al., 2008
• Decreased labile soil
organic matter
• Frey et al 2008, Melillo
et al 2011
• Decreased fungal
biomass
• Frey et al 2008
Δ(Htd
–
Ctl)
Soil
CO
2
flux
(g
CO
2
-C
m
-2
y
-1
)
Melillo et al., Science 2017

16. Soil C loss has been discontinuous and non-linear over
30 years of warming
Δ(Htd
–
Ctl)
Soil
CO
2
flux
(g
CO
2
-C
m
-2
y
-1
)
Melillo et al., Science 2017

17. “Our biogeochemical and molecular
observations suggest that warming causes
cycles of soil carbon decay punctuated by
periods of structural and functional
changes in the microbial community.”
Melillo et al., Science 2017

18. Acknowledgements
Photo by Audrey Barker-Plotkin
• Mallory Choudoir, Achala Narayanan, Ashley Eng,
Rachel Simoes, Alon Efroni, Vedang Diwanji
• Will Werner, Michael Bernard, Jerry Melillo
(Marine Biological Laboratory)
• Serita Frey, Mel Knorr, Stuart Grandy (UNH)
• UMass Genomics Resource Laboratory, Ravi
Ranjan (UMass)
• National Science Foundation CAREER award
program DEB-1749206
• We live and work on Nonotuck land, neighboring
Indigenous nations: the Nipmuc and the
Wampanoag to the East, the Mohegan and
Pequot to the South, the Mohican to the West,
and the Abenaki to the North.

19. • Sequence-based approaches
• Detect changes at the community level
• Most organisms resist cultivation
• Cultivation-based approaches
• Detect changes at the organismic level
• Only way to test adaptation
• Genomics, sequencing whole genomes
• Transcriptomics, sequencing RNA transcripts
• Proteomics, sequencing proteins
• Meta-, sequencing from mixed communities
• MAGs, Metagenome-assembled genomes
• …aka ‘Omics
Crick, Nature 1970
Thermus thermophilus
small subunit (16S)
ribosomal RNA.
Proteins in blue,
rRNA in orange.
How do we find out what the
microbes are doing?

20. DeAngelis et al., Frontiers Microbiol. 2015
Stress reduced fungal biomass, but not bacterial.
Some phyla even increased in abundance!
20

21. DeAngelis et al., 2015
Bacterial
community
composition
(top)
&
community
diversity
(bottom)
Soil carbon loss with warming is associated with
small changes in community structure.
… likely due to
environmental filtering
- We assume the
environment has
changed, and caused
communities to change

22. Long-term
warmed soils
have more
carbohydrate
degrading genes
22
Pold et al., 2016

23. Acclimation
versus adaptation
• Ecology vs evolution
• Fast vs slow
• Temporary vs permanent
Chapman T&F 2008
23

24. Adaptation is encouraged with environmental stress.
… warming decreased soil organic matter quantity
Organic horizon
Mineral soil
Pold et al., SBB 2017
25-30% C
1-1.5% N
6-9% C
0.25-0.4% N
Grace
Pold

25. Pold et al., SBB 2017
Organic horizon
Mineral soil
Adaptation is encouraged with environmental stress.
… warming decreased soil organic matter quality

26. Acclimation versus adaptation
• Evidence for acclimation
• Change in community structure
• Changes in gene expression of existing microbes
• Evidence for adaptation
• Increased stress (necessary but not sufficient!)
• Different traits in organisms from stressed environments
• Genomic signatures of organisms from stressed environments
• Increased fitness due to adaptive traits
• Both are happening at the same time, so we’re looking for direct,
genomic adaptation!
26

27. • Sequence-based approaches
• Detect changes at the community level
• Most organisms resist cultivation
• Cultivation-based approaches
• Detect changes at the organismic level
• Only way to test adaptation
Crick, Nature 1970
How do we find out what the
microbes are doing?

28. Workflow for building and analyzing the culture collection

29. Workflow for building and analyzing the culture collection
1. Collect soils from the experiment, separating the overlying organic
horizon from the underlying mineral soils
2. (Optional) Incubate soils under restrictive conditions, or otherwise enrich
for certain types of organisms.
3. Extract cells into an isotonic solution like phosphate-buffered saline (PBS)
so that they do not lyse in the transfer.
4. Dilute the extract down to apply 10-100 cells per plate, and spread the
solution onto plates filled with agar media.
5. Pick colonies and genotype by sequencing the 16S ribosomal RNA gene.
6. Measure the physiology to understand how warming has affected the
physiology.
7. Use physiology, genotype, and other information to choose which
isolates to sequence the genomes.

30. This year’s isolates are isolated from soils archived over
the course of the field warming experiment
Δ(Htd
–
Ctl)
Soil
CO
2
flux
(g
CO
2
-C
m
-2
y
-1
)
Melillo et al., Science 2017
1998 2002 2013 2020
• All organisms were
isolated in 2021
and 2022
• Orange arrows
show when soil
was collected &
dried
• The idea is that
these organisms
represent spores
deposited at
different times
• Could this permit a
look back in
evolutionary time?

31. The Chronic Warming Culture Collection
• >800 individuals from control
and heated soils representing 7
phyla in 42 families
• Phylogenetic tree at right shows
283 of the isolates
• Color bar on the outside denotes
origin of isolates
• Red bar isolates are from heated
plots
• Blue bar isolates are from control
plots or just outside of the
experimental plots
• Branches are color coded by
phylum
31

32. Many microbial traits
show phylogenetic
conservation
• This phylogenetic tree
of bacteria includes
ribosomal operon copy
number mapped as
traits
Kembel et al., PLOS Comp Biol 2012

33. Testing whether traits from two groups of
organisms are different
• Most statistical comparisons (like t-test) assume that observations are observed independently.
• Traits associated with individuals are NOT independent… in fact, phylogenetic trees are model
depictions describing their relationship
• Ignoring phylogeny in comparative analysis assumes star phylogeny
33
Garland & Carter, 1994

34. Purvis & Rambaut, 1995
METHODS: Phylogenetic Group Comparison
Fig. 1. An illustration of the pitfalls posed by comparative data. Six species are related as shown in (a).
Analyses treating the species values as independent can find apparently significant correlations
between Y and X even when there is no relationship within either clade (b) and can miss correlations
that are strong within each clade (c). After Gittleman and Luh (1992)

35. Phylogenetically independent contrasts
• The logic of the method is to use
phylogenetic information to
transform the original tip data into
values that are statistically
independent and identically
distributed.
• Brownian motion is the assumed
model of trait evolution
• Each tip is a species or set of species
• Tip data is a value per species or a
mean value of the species set
35
https://en.wikipedia.org/wiki/Phylogenetic_comparative_methods

36. Altered carbohydrate-degradation potential
Chronic warming is associated with
increased potential to degrade
• cellulose (CMC)
• Xylan
• Not chitin
Pold et al., AEM 2016

37. Warming is associated with …
temperature sensitivity of growth
• Alphaproteobacteria grown
at temperatures 24 - 39oC
• Gompertz model fit to
extract growth parameters
A. Eng, in prep
37

38. Warming is not? associated with …
temperature sensitivity of growth
• Like soils, microbes exposed to
warming show lower
temperature sensitivity of
growth
Growth rate per change degrees C
A. Eng, in prep
38

39. Model soils mimic soil living for microbes
• Model soils =
• Minerals (Sand, Clay)
• Simple organics (sugar, nutrients)
• Actinobacteria

40. **
*
Warming is associated with…
increased drought tolerance
A. Narayanan, in prep
The lower the water content, the larger
the effect of warming on growth.

41. Addition and
loss of
species
Changes in community
composition and
structure
Genetic changes
(adaptation)
Generations of long-term warming
Fast response Slower response
Adapted from Bang et al. (2018)
Environmental filtering and adaptation are likely
co-occurring across different temporal scales.

42. The Chronic
Warming Culture
Collection
42
Collect soil samples
from control and
heated plots
Targeted enrichment
of microbial taxa
Whole genome
MinION sequencing
Mallory
Choudoir

43. Pangenomes can identify genomic signatures
of adaptation to long-term warming
McInerney et al. (2017)
43

44. Pangenomes can identify genomic signatures
of adaptation to long-term warming
McInerney et al. (2017)
44

45. • Whole genome phylogeny using
concatenated ribosomal proteins
• 78 soil bacteria isolated from HF
long-term warming experiment
• 3 Phyla (Actinobacteria,
Alphaproteobacteria,
Betaproteobacteria)
• 5 Genus (Kitasatospora,
Streptomyces, Bradyrhizobium,
Rhizobium, Ralstonia)
Comparative genomics
M. Choudoir, in prep

46. Pangenome analysis (n=78)
M. Choudoir, in prep

47. Codon usage bias increases with warming
M. Choudoir, in prep
47

48. Codon usage bias
• A measure of an
organism’s
preference for
one codon &
tRNA for an
amino acid
48

49. Codon usage bias
• The genetic code
is redundant, with
synonymous
codons– different
codons which
encode for the
same amino acid
49

50. Codon usage bias is associated with lifestyle
50
https://doi.org/10.1186/gb-2011-12-10-r109

51. Lecture Learning Goals
• Explain how the Earth’s climate is changing, and describe the
symptoms of climate change.
• Define soil, and explain why it is an important element in healthy
ecosystems as well as a possible climate solution.
• Describe the long-term warming field experiment ongoing at the
Harvard Forest in central Massachusetts.
• Explain the evidence for bacterial adaptation to long-term warming.
• Describe the origin of the genomes that you are going to work with
for your capstone projects, and describe how genomes were chosen
for genome sequencing.
51