Characterising the CRISPR immune system in Archaea

219 Pages·2012·14.53 MB·English

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￿D￿a￿n￿i￿sh￿￿A￿￿rc￿h￿ae￿a￿C￿e￿n￿t￿re￿￿￿￿￿
http://dac.molbio.ku.dk
2012, September
Shiraz A. Shah: Characterising the CRISPR immune system in Ar-
chaea using genome sequence analysis, Ph. D. thesis, 2012, Septem-
ber.
:
http://dac.molbio.ku.dk/index.php?page=shirazcv
￿￿￿￿￿￿￿
:
[email protected]
￿-￿￿￿￿
ThisPh. D.thesisisbasedonworkcarriedoutbetweenMay2007
andJuly2012inDanishArchaeaCentre,UniversityofCopenhagen
(http://dac.molbio.ku.dk/).
ABSTRACT
Archaea, a group of microorganisms distinct from bacteria and
eukaryotes,areequippedwithanadaptiveimmunesystemcalled
the CRISPR system, which relies on an RNA interference mech-
anismtocombatinvadingvirusesandplasmids. Usingagenome
sequence analysis approach, the four components of archaeal
genomic CRISPR loci were analysed, namely, repeats, spacers,
leaders and cas genes. Based on analysis of spacer sequences it
was predicted that the immune system combats viruses and plas-
mids by targeting their DNA. Furthermore, analysis of repeats,
leaders and cas genes revealed that CRISPR systems exist as dis-
tinct families which have key differences between themselves.
Closely related organisms were seen harbouring different CR-
ISPRsystems,whilesomedistantlyrelatedspeciescarriedsimilar
systems, indicating frequent horizontal exchange. Moreover, it
was found that cas genes of Type I CRISPR systems could be di-
vided into functionally independent modules which occasionally
exchange to form new combinations of Type I systems. Further-
more, Type III systems were found to be genomically associated
with various combinations of accessory genes which may play a
role in functionally extending the activity of the Type III inter-
ference complexes. This dynamic nature of the CRISPR immune
systemsmaybeaprerequisitefortheircontinuedefficacyagainst
the ever changing threats they protect their hosts from.
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SUMMARY
Archaea comprise a group of microorganisms distinct from both
bacteria and eukaryotes. These organisms are equipped with an
adaptive immune system against invading viruses and plasmids.
The immune system works by taking up DNA from a virus, and
savingitonthehost’sownchromosomeasatemplatetoproduce
interference RNA. The RNA recognises the virus the next time
it infects and signals the degradation of its genetic material. All
the components of this system are encoded on the chromosomes
of the organisms, and by looking into these components using
genome sequence analysis, a number of insights were gained.
It was found that the immune system kills viruses by targeting
their DNA, first and foremost. Furthermore, different archaea
have different variants of the immune system, and most archaea
harbour several variants at the same time, probably to aid them
in targeting different types of viruses. The systems themselves
are composed of independent modules which are responsible for
different stages of the immune response. By combining the mod-
ulesinvariouscombinationsandextendingthemwithadditional
components as well as exchanging them with other archaea, the
organisms ensure that their immune systems are fit to handle
diverse and continuously evolving threats.
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SAMMENFATNING
Arkæa udgør en gruppe af organismer som er forskellige fra
både bakterier og eukaryoter. De er udstyrret med et adaptivt
immun system mod invaderende vira og plasmider. Immunsy-
stemet virker ved at optage virussens DNA, som bliver gemt i
værtens eget kromosom for derved at blive brugt som skabelon
til fremstilling af interferens RNA. RNA’et genkender virussen
næste gang den inficerer, og signalerer derved for nedbrydelsen
af virussens genetiske materiale. Alle immunsystemets kompo-
nenter er indkodet i organismernes DNA, og ved at undersøge
komponenterne gennem genom sekvens analyse blev der gjort
en række opdagelser. Vi fandt ud af at immunsystemet dræber
vira ved først og fremmest at angribe deres DNA. Derudover
har forskellige arkæa forskellige varianter af immun systemet, og
de fleste arkæa besidder flere af varianterne på én gang, hvilket
sandsynligvis hjælper dem med at kunne tackle forskellige typer
vira. Selve immunsystemerne består af uafhængige moduler som
hver især står for forskellige stadier af immun reaktionen. Ved at
kombinere modulerne i forskellige kombinationer eller udvide
dem med yderligere komponenter samt at ombytte dem med
andre arkæa, sikrer organismerne sig at deres immun system
er opdateret til at kunne modstå de forskelligartede trusler som
hele tiden udvikler sig.
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PREFACE
The work presented in this thesis was carried out at the Dan-
ish Archaea Centre at the Department of Biology, University of
Copenhagen from May 2007 to July 2012 under the supervision
of Professor Roger A. Garrett.
The initial objective of the Ph.D. study was to characterise
the CRISPR immune system in Sulfolobus species using computa-
tionalmethods,especiallycomparativegenomesequenceanalysis.
Sulfolobus species have been extensively studied with regard to
the viruses and plasmids which infect them, with many genome
sequences available of hosts as well as their extrachromosomal
elements. Furthermore, Sulfolobus species harbour extensive and
diverse CRISPR immune systems. Thus there was more than
enough data to begin the analyses. After exhausting the possible
ways of analysing CRISPR spacer, repeat, leader and cas gene
sequences from Sulfolobales, the analyses were extended to the
rest of the available archaeal genomes in collaboration with Dr.
Gisle A. Vestergaard starting July 2010. Gisle worked with me on
theprojectuntilOctober2011afterwhichIovertookit. Extending
the study to other archaea proved fruitful, but was also a big
mouthful, and these analyses are still in the process of being
completed. Preliminary results have, however, been included in
this thesis, to which especially the last part is dedicated.
TheresultsfromthisPh.D.studyhavebeenpublishedthrough-
out many individual research papers, which are all enclosed,
and most of which have multiple co-authors. Therefore the ex-
tent of my own contributions to each of these papers have been
stipulated on the sheet preceding every paper.
For the sake of clarifying the extent of my collaboration with
Dr. Gisle A. Vestergaard and its influence on what is presented
in this thesis, he was deeply involved in all work concerning
the classification of archaeal cas genes into separate functional
modules(aCas, iCas, etc.), andtheclassificationofiCmrmodules
into 5 families, A through E. In these studies our workload
was more or less equal. Some aspects of this work is already
published while others remain. As for the analysis of archaeal
CRISPR repeats and leaders, as well as the definition of iCmr
accessory genes, these analyses were conducted by my myself
after Gisle left the project and are still unpublished.
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ACKNOWLEDGEMENTS
First and foremost I’d like to thank my supervisor, Professor
RogerA.Garrett. Wehadmanylong,inspiringdiscussions. Also,
with Roger I saw how experience and wisdom go hand in hand.
But most importantly I want to thank him for having patience
and maintaining his trust in me during the challenges which
were faced.
During my Ph.D. I made friends. Chandra, Gisle, Chao and
Ling. Four friends in four years. It’s difficult not to be thankful
for that.
Also, I had the privilege of working in a lab full of absolutely
terrific people. I can’t think of a single exception. Despite many
ofushavingtodealwithourownday-to-daychallenges,between
ustherewasagenuinelypositiveandunderstandingatmosphere.
When comparing with most other workplaces you realise that
this is the kind of thing you mustn’t take for granted.
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1 1
CONTENTS
1.1 The tree of life 1
￿1￿.2￿￿￿A￿r￿ch￿￿a￿e￿a￿- the third domain of life 3
1.2.1 Defining characteristics of Archaea 3
1.2.2 Archaeal diversity: Ecology, taxonomy and
genomics 8
1.3 Sulfolobus, a crenarchaeal model organism 12
1.3.1 Sulfolobus genomics 14
1.3.2 Sulfolobales viruses and plasmids 16
1.4 CRISPR: Adaptive immunity in archaea and bac-
teria 19
1.4.1 A history of the research 19
1.4.2 CRISPR bioinformatics 21
2 27
2.1 Spacers 27
￿￿￿￿2￿￿.1￿.1 A note on nucleotide vs. amino acid spacer
matches 28
2.2 Repeats 28
2.3 Leaders 29
2.4 cas genes and CRISPR genomic context 31
2.4.1 Evidence for iCmr accessory genes 32
3 35
4 39
5 ￿￿￿￿￿￿￿￿￿￿ & ￿￿￿4￿1￿￿￿￿￿￿￿￿
￿5.￿1￿￿P￿￿ap￿e￿￿r￿1 42
￿5.￿2￿￿￿P￿a￿p￿e￿￿r￿2￿ 52
5.3 Paper 3 59
5.4 Paper 4 74
5.5 Paper 5 89
5.6 Paper 6 103
5.7 Paper 7 116
5.8 Paper 8 126
5.9 Paper 9 134
5.10 Paper 10 146
5.11 Book chapter A 155
5.12 Book chapter B 176
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