Molecular microbial ecology of Antarctic lakes Sheree Yau

244 Pages·2013·2.24 MB·English

Molecular microbial ecology of Antarctic lakes
Sheree Yau
A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy
School of Biotechnology and Biomolecular Sciences
Faculty of Science
University of New South Wales, Australia
February, 2013
PLEASE TYPE
THE UNIVERSITY OF NEW SOUTH WALES
Thesla/Dissertation Sheet
Surname or Family name: Yau
First name: Sheree Other namels:
Abbreviation fcx degree as given in the University calendar· PhD
School: Biotechnology and Biomolecular Sciences Faculty: Faculty of Science
Tltte: Molecular microbial ecology of Antarctic lakes
Abs1Tac:t 350 words maximum: (PLEASE TYPE)
The Vestfold Hills is a coastal Antarctic oas1s, a rare ic&-free region containing a high density of meromictic (permanently stratifice<l) lakes. These
lakes are ideal model ecosystems as their microbial communities exist along physico-chemical gradients, allowing populations tc) be correlated
with geochemical factors. As extensive historic, physico-chemical and biological datasets exist for Ace Lake and Organic Lake. two marine-derived
meromictic lakes, they were chosen as study sites for molecular-based analysis·o f their microbial communities.
Analysis of genetic material randomly sequenced from the environment (metagenomlcs) was performed to determine taxonomic composition and
metabolic potential. To support metagenomic inferences, methods were developed for performing microscopy on lake water samples and for the
identification of proteins from the environment (metaproteomics). Metaproteomic analysis Indicated active community members, while
microbial/viral abundances were determined by microscopy. An integrative approach combining metagenomic, metaproteomic and physico
chemical data enabled comprehensive descriptions of the lake ecosystems.T his included the Identification of taxa not previously known to inhabit
the lakes and determination of biogeochemical cycles.
A complete genome was reconstructed of a member of the virophage viral family and near complete genomes of phycodnaviruses. The virophage
likely preys on phycodnaviruses that infect eucaryotic phytoflagellates. A model of virophag&-phycodnavirus-algae population d)lflamics predicted
the presence of a virophage Increases the frequency of algal blooms and thus overall nutrient release. Virophage signatures wetre detected in
other aquatic envcronments indicating they play a previously unrecognised role in other environments. In Organic Lake, genes a:;sociated with
heterotrophic bacteria involved in OMSP cleavage. photoheterotrophy, llthoheterotrophy and nitrogen remineralisallon were abutndanl, indicating
these processes are adaptations to nutnent constraints. Photo-and lithoheterotrophy enables carbon to be used for bios)lflthesis rather than
energy generation thereby conseNing carbon in the lake, while recycling of nitrogen limits its loss. DMSP appears to be significc10t carbon and
energy source and also the origin of high OMS in Organic Lake. These molecular-based discoveries shed light on the rote of previously
unrecognised taxa and metabolic processes In unique Antarctic lake environments.
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I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this Is app11lcable to doctoral
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contribution made to the research by others, with whom I have worked at
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archive and to make available my thesis or dissertation in whole or part in the
University libraries in all forms of media, now or here after known, subject to the
provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent
rights. I also retain the right to use in future vxorks (such as articles or books) all
or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in
Dissertation Abstract International (this is applicable to doctoral theses only).
I have either used no substantial portions of copyright material in my thesis or I
have obtained permission to use copyright material; where permission has not
been granted I have applied/will apply for a partial restriction of the digital copy of
my thesis or dissertation.'
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Abstract
The Vestfold Hills is a coastal Antarctic oasis, a rare ice-free region containing
a high density of meromictic (permanently stratified) lakes. These lakes are ideal
modelecosystemsastheirmicrobialcommunitiesexistalongphysico-chemicalgra-
dients,allowingpopulationstobecorrelatedwithgeochemicalfactors. Asextensive
historic, physico-chemical and biological datasets exist for Ace Lake and Organic
Lake, two marine-derived meromictic lakes, they were chosen as study sites for
molecular-based analysis of their microbial communities.
Analysisofgeneticmaterialrandomlysequencedfromtheenvironment(metage-
nomics)wasperformedtodeterminetaxonomiccompositionandmetabolicpoten-
tial. To support metagenomic inferences, methods were developed for performing
microscopy on lake water samples and for the identification of proteins from the
environment (metaproteomics). Metaproteomic analysis indicated active commu-
nity members and processes, while microbial/viral abundances and morphology
weredeterminedbymicroscopy. Anintegrativeapproachcombiningmetagenomic,
metaproteomic and physico-chemical data enabled comprehensive descriptions of
the lake ecosystems. This included the identification of taxa not previously known
to inhabit the lakes and determination of biogeochemical cycles.
A complete genome was reconstructed of a member of the newly described
virophage viral family and near complete genomes of phycodnaviruses. The vi-
rophagelikely‘preys’onphycodnavirusesthatinfecteucaryoticphytoflagellates. A
model of virophage–phycodnavirus–algae population dynamics predicted the pres-
enceofavirophageincreasesthefrequencyofalgalbloomsandthusoverallnutrient
release. Virophage signatures were detected in other aquatic environments indi-
cating they play a previously unrecognised role in other environments. In Organic
Lake, genes associated with heterotrophic bacteria involved in DMSP cleavage,
photoheterotrophy, lithoheterotrophy and nitrogen remineralisation were abun-
dant, indicating these processes are adaptations to nutrient constraints. Photo-
and lithoheterotrophy enables carbon to be used for biosynthesis rather than en-
ergy generation thereby conserving carbon in the lake, while recycling of nitrogen
limitsitsloss. DMSPapppearstobesignificantcarbonandenergysourceandalso
the origin of high DMS concentrations in Organic Lake. These molecular-based
discoveries shed light on the role of previously unrecognised taxa and metabolic
processes in unique Antarctic lake environments.
ii
Acknowledgements
Extra special thanks go to my supervisor, Rick Cavicchioli, and my co-supervisor,
Federico Lauro. Without their support, useful discussions, editorial assistance and
much needed prods along throughout the course of my PhD, I certainly would not have
gotten this done or come anywhere near as far. I am very grateful for the time, energy,
patience and hard work that was put into me. I have learnt a lot!
Thanks to all those involved in the logistics of Antarctic research and especially
sample collection. Expeditioners who collected samples used in this thesis were Rick
Cavicchioli, Federico Lauro, Torsten Thomas, Mark Brown, Jeff Hoffman and John
Rich. Thanks to Nico Wanandy for the logistic support at the UNSW end and folks
fromtheAustralianAntarcticDivision. SpecialthankstoJohnGibsonforcontributing
his wealth of knowledge about the lakes.
Thanks to the rest of the lab-family from whom I have also learnt a lot and who
have been wonderful friends to work with. Tim Williams, Mark Brown and Matt
DeMaere deserve a special mention for their help, advice and support on the projects.
Very special thanks go to David Wilkins and Suhaila Mohd. Omar who have worked
alongside with me as fellow PhD companions and without whom I would have been
lost. Thanks to Jodi Richards, Tim Charlton, Sohail Siddiqui and Haluk Ertan for
the invaluable lunchtime support group meetings. Thanks to the past PhD students
from the group, Charmaine Ng, Dominic Burg and Davide De Francisci, who left big
footprints for me to follow. Thanks to my extended university family, Jo and Malu,
for being my much needed tea and lunch break companions, and Kylie and Pan, for all
the mind-expanding biology discussions.
To my at-home science family, thanks for being there as my constant, private sup-
port team and my human/non-human pack to run through life with. Very special
thanks to Amy Cain, who has been through the whole gamut of education with me
until this point where all the official avenues of higher learning have been exhausted,
and to Laura Nolan, who has ended up being as good as a de facto wife as well as thesis
sister to me. Thanks to Julia Suurbach, Meaghan Kukulj, Sissy Reyes, Sophia Slavich
and Tim L Williams for being such good friends and inspiring people to be around.
Thanks to Rosko, Mutley, Puggy, Calypso, Icarus, Nakita I and II for the much needed
walks, fly-arounds and just hanging around as part of the family.
A very special thanks to my genetic family without whom I definitely would not
havebeenheretodothis. Thanksmum, popandJerson, Ioweyoueverything. Thanks
Indy (I know we are only very distantly related really, but it doesn’t matter).
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List of Publications
In publications arising from my PhD work, my supervisor Prof Ricardo Cavicchioli and
my co-supervisor Dr Federico Lauro were involved in the research design and editing
of the manuscripts. Where versions of published material, or material submitted for
publication appears in this thesis, details of the contributions made by myself and
others precede it.
• Sheree Yau, Federico M. Lauro, Timothy J. Williams, Matthew Z. DeMaere,
Mark V. Brown, John Rich, John A.E. Gibson, Ricardo Cavicchioli. Strategies
of carbon conservation and unusual sulfur biogeochemistry in a hypersaline lake.
The ISME Journal (submitted), 2013.
• KhawarS.Siddhiqui,TimothyJ.Williams,DavidWilkins,ShereeYau,Michelle
A.Allen,MarkV.Brown,FedericoM.Lauro,RicardoCavicchioli. Psychrophiles.
Annual Review of Earth and Planetary Sciences(doi: 10.1146/annurev-earth-040610-
133514), 2013.
• David Wilkins, Sheree Yau, Timothy J. Williams, Michelle Allen, Mark V.
Brown, Matthew Z. DeMaere, Federico M. Lauro and Ricardo Cavicchioli. Key
Microbial Drivers in Antarctic Aquatic Environments.
FEMS Microbiology Reviews (doi:10.1111/1574-6976.12007), 2012.
• Sheree Yau and Ricardo Cavicchioli. Microbial communities in Antarctic lakes:
Entirely new perspectives from metagenomics and metaproteomics.
Microbiology Australia 32:157–159, 2011.
• Sheree Yau, Federico M. Lauro, Matthew Z. DeMaere, Mark V. Brown, Torsten
Thomas, Mark J. Raftery, Cynthia Andrews-Pfannkoch, Matthew Lewis, Jeffrey
M. Hoffman, John A. Gibson and Ricardo Cavicchioli. Virophage control of
antarctic algal host–virus dynamics.
Proceedings of the National Academy of Sciences USA 108:6163–6168, 2011.
• Federico M. Lauro, Matthew Z. DeMaere, Sheree Yau, Mark V. Brown, Char-
maine Ng, David Wilkins, Mark J. Raftery, John A.E. Gibson, Cynthia Andrews-
Pfannkoch, Matthew Lewis, Jeffrey M. Hoffman,Torsten Thomas and Ricardo
Cavicchioli. An integrative study of a meromictic lake ecosystem in Antarctica.
The ISME Journal 5:879–895, 2011.
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Contents
1 General introduction 1
Co-authorship statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Antarctic lakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 The Vestfold Hills . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Insights from molecular studies of Antarctic lakes . . . . . . . . . . . . . 3
1.3.1 Bacterial diversity: adaptation to unique physical and chemical
conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.2 Archaea: methanogens and haloarchaea . . . . . . . . . . . . . . 7
1.3.3 Eucarya perform multiple ecosystem roles . . . . . . . . . . . . . 8
1.4 Integrative studies to derive whole ecosystem function . . . . . . . . . . 9
1.4.1 A single gene approach . . . . . . . . . . . . . . . . . . . . . . . 9
1.4.2 ‘-omics’ approaches . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5 History of research on Ace and Organic Lakes . . . . . . . . . . . . . . . 12
1.6 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2 Development of methods to complement metagenomic sequencing for
an integrative study of Ace Lake 15
Co-authorship statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.1 Ace Lake samples. . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.2 DNA extraction, sequencing and data cleanup . . . . . . . . . . 19
2.3.3 Metagenomic DNA assembly and annotation . . . . . . . . . . . 20
2.3.4 Epifluorescence microscopy . . . . . . . . . . . . . . . . . . . . . 20
2.3.5 Protein extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3.6 1D-SDS PAGE and LC-MS-MS . . . . . . . . . . . . . . . . . . . 21
2.3.7 Metaproteomic mass spectra analysis . . . . . . . . . . . . . . . . 21
2.4 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.4.1 Development of an epifluorescence microscopy method . . . . . . 22
2.4.2 Community stratification supported by cell and VLP densities . 24
2.4.3 Development of a metaproteomic mass spectra analysis workflow 26
2.4.4 Insights from the metaproteomic analysis of Ace Lake . . . . . . 31
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