4. Genome-based comparative analyses of Antarctic and temperate species of Paenibacillus

154 Pages·2015·3.62 MB·English

http://researchspace.auckland.ac.nz

ResearchSpace@Auckland

Copyright Statement

The digital copy of this thesis is protected by the Copyright Act 1994 (New
Zealand).

This thesis may be consulted by you, provided you comply with the provisions of
the Act and the following conditions of use:

• Any use you make of these documents or images must be for research or
private study purposes only, and you may not make them available to any
other person.
• Authors control the copyright of their thesis. You will recognise the
author's right to be identified as the author of this thesis, and due
acknowledgement will be made to the author where appropriate.
• You will obtain the author's permission before publishing any material from
their thesis.

To request permissions please use the Feedback form on our webpage.
http://researchspace.auckland.ac.nz/feedback

General copyright and disclaimer

In addition to the above conditions, authors give their consent for the digital copy
of their work to be used subject to the conditions specified on the Library Thesis
Consent Form and Deposit Licence.
Genomic and phenotypic insights
into the ecology of Antarctic soil bacteria

Melissa Denis Dsouza

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of
Philosophy, University of Auckland, 2015.
Abstract
Antarctic soils represent a unique environment, characterised by extremes of temperature,
salinity, elevated UV radiation, low nutrient and low water content. Despite the harshness of this
environment, members of 15 bacterial phyla have been identified in soils of the Ross Sea
Region. However, the survival mechanisms and ecological roles of Antarctic bacteria are largely
unknown. The aim of this thesis is to improve our understanding of the physiological adaptations
that give bacterial species their ability to grow and survive in the harsh, nutrient-depleted
Antarctic soil environment. For this, three Antarctic strains of the novel bacterial species
Paenibacillus darwinianus, isolated in this study, and seven Antarctic members of the bacterial
genus Arthrobacter were investigated by whole genome sequencing and phenotypic
characterisation. Genome-based comparative analyses with temperate, soil-dwelling counterparts
were performed to investigate whether these 10 isolates owe their resilience to substantial
genomic changes. Genome analysis of all 10 isolates revealed several genes for signal
transduction, sigma factors, the production and/or uptake of compatible solutes, the removal of
reactive oxygen species and those for the maintenance of optimal membrane fluidity. However,
these genes were also identified in genomes from cold-adapted bacteria and archaea as well as in
genomes from temperate, soil-dwelling counterparts. This suggests that these physiological traits
are not unique to cold soil environments but are beneficial for growth and survival in a range of
soil conditions. Comparative analyses also provided some evidence for genome content scaling
in four of the seven Arthrobacter isolates and in all strains of P. darwinianus. In the Antarctic
soil environment, complex C sources are largely absent, making a fixed range of labile organic
residues the predominant source of C and N. As a result, the maintenance of reproductive
efficiency, promoted by smaller genomes, is more crucial for growth and survival than the
maintenance of metabolic complexity, promoted by larger genomes. Overall, this study has
produced cultured isolates that represent a platform for more in-depth study of novel traits for
adaptation to the Antarctic soil environment or for the discovery of novel cold-adapted enzymes
for biotechnological uses. In addition, the genome sequences produced are a resource for further
investigations into the expression of physiological attributes that enable survival under extreme
conditions and the selection processes that affect prokaryotic genome evolution.
i
Acknowledgements
I would like to start by thanking my three supervisors Mike Taylor, Sue Turner and Jackie
Aislabie. It was their endless support, guidance and good counsel that kept me going. Special
thanks to Jackie who gave me the very unique opportunity to go down to Antarctica and to Mike
a.k.a Dr Taylor for welcoming me back to the Taylor group and “cough” not calling me names
such as „traitor‟ or D‟Loser‟ or „D‟Poosa‟, or for throwing paper balls at me. A big thank you to
the Taylor-Turner Lab groups who‟ve been supportive and have on several occassions listened to
my rants about failed experiments, computer programs that won‟t work and bacteria that refuse
to grow. I would also like to thank them for “cough” not making any jokes on „Paenibacillus
darwininanus‟ and letting it go.

Thanks to Dave for all his help with linux-based computer programs, his general good counsel
and for being cookie-accquistion officer, Racy for the early mornings, gym sessions, baking and
cups of tea, Kristi for lab advice, more tea and supervised baking, Manpreet for speaking hindi,
advice on everything and for being awesome!, Anna for teaching me how bacteria grow and
Gavin Lear who gave me advice on all soil-related matters. Thanks to the newer Taylor lab
ii
members Brett, Carmen, Elahe and Michael Michael for being there for a chat and for being less
jaded by research. A special thanks to Brett and Simiee for their general awesomeness.

A big thank you to Ruth for all the autoclaving (of which there was so much!) and for also
participating in my rants, Marlene for being my knight in shining armour for all admin- and
reagent ordering-related jobs. My thesis would have been impossible with you. Special thanks to
Peter Tsai for his genome assembly and analysis-related advice. Thanks to all members of the
Microbiology Seminar Group who‟ve sat through my presentations and given me advice, Adrian
Turner for all his assistance with electron microscopy, Bob Anderson and Roger van Ryn for
their assistance with the gamma-irradiator, Judy O‟Brien and Sue Skelly for their help with every
other administrative hurdle. A very big thank you to Antarctica NZ and the staff at Scott Base for
getting me down to the ice and back, safely, providing assistance with sampling and making it a
trip I‟d never forget!

I would also like to thank my flatties/friends, Vania for listening when times were tough and
days long and for yet more tea, Alice for introducing me to Pims and Buffy-The Vampire Slayer,
Fiona for introducing me to better TV shows and, Michael a.k.a mini Mike for listening to my
endless complaints on everything, cooking, reading all my drafts and enabling my rum and coke
drinking habits. A big thanks to Greer, Bob, Amy, Alasdair and the kids Caleb and Jacob for
being so loving and understanding and for keeping me sane.

Finally, thank you to my mum, dad and brother for being patient and supportive. Thank you for
believing in me, I could not have done this without you.

iii
Table of Contents
Abstract ..................................................................................................................... i
Acknowledgements.................................................................................................. ii
List of Figures ......................................................................................................... vi
List of Tables ........................................................................................................ viii
1. General Introduction ........................................................................................... 9
1.1 Antarctica .............................................................................................................................. 9
1.1.1 The soil environment ................................................................................................... 12
1.2 Life in Antarctica ................................................................................................................ 16
1.2.1 Bacterial diversity and distribution in Antarctic soils.................................................. 18
1.3 Adaptations to Antarctic soil environments ........................................................................ 19
1.3.1 Responding to the cold: Temperature sensors ............................................................. 20
1.3.2 Responding to osmotic stress: Accumulation of compatible solutes ........................... 22
1.3.3 Responding to elevated UV radiation: The DNA damage response ............................ 24
1.3.4 Responding to multiple stresses by pigment production ............................................. 27
1.3.5 Responding to nutrient limitation ................................................................................ 28
1.3.6 The cold shock response .............................................................................................. 30
1.4 Studying adaptation by whole genome sequencing ............................................................ 31
1.5 Research aims ..................................................................................................................... 33
1.6 References ........................................................................................................................... 36
2. Genomic and phenotypic insights into the ecology of ....................................54
Arthrobacter from Antarctic soils .........................................................................54
2.1 Introduction ......................................................................................................................... 56
2.2 Results and Discussion ....................................................................................................... 58
2.2.2 Genome analyses ......................................................................................................... 60
2.2.3 Genomic traits linked to environmental stress-related adaptation ............................... 68
2.2.4 Phenotypic characterisation of Antarctic Arthrobacter strains .................................... 75
2.3 Conclusions ......................................................................................................................... 78
2.4 Materials and Methods ........................................................................................................ 79
iv
2.4.1 qPCR analysis .............................................................................................................. 79
2.4.2 Phylogenetic analyses of Arthrobacter from soils of the RSR .................................... 81
2.5 Acknowledgements ............................................................................................................. 84
2.6 List of abbreviations ........................................................................................................... 84
2.7 References ........................................................................................................................... 85
3. Paenibacillus darwinianus sp. nov., isolated from gamma-irradiated
Antarctic soil ...........................................................................................................94
4. Genome-based comparative analyses of Antarctic and temperate species of
Paenibacillus .........................................................................................................109
4.1 Introduction ....................................................................................................................... 111
4.2 Results and Discussion ..................................................................................................... 113
4.2.1 Genome overview ...................................................................................................... 113
4.2.2 General genome comparisons .................................................................................... 116
4.2.3 Genomic features linked to survival in stressful conditions ...................................... 119
4.3 Conclusions ....................................................................................................................... 127
4.4 Materials and Methods ...................................................................................................... 128
4.4.1 Habitat and isolation strategy..................................................................................... 128
4.4.2 DNA extraction and sequencing ................................................................................ 129
4.4.3 De novo assembly, annotation and comparative analyses ......................................... 129
4.4.4 Genome Submission .................................................................................................. 130
4.5 Acknowledgements ........................................................................................................... 130
4.6 References ......................................................................................................................... 131
5. General discussion ............................................................................................141
5.1 Genome content ................................................................................................................ 142
5.2 Genome content scaling .................................................................................................... 144
5.3 Bacterial cultivation .......................................................................................................... 145
5.4 Future directions ............................................................................................................... 145
5.5 References ......................................................................................................................... 148
6. Appendices (included on CD) .........................................................................152
v
List of Figures
Figure 1.1: Map of Antarctica showing the main geographic features. Source: Landsat Image
Mosaic of Antarctica (http://lima.nasa.gov/pdf/A3_overview.pdf). ............................................. 10
Figure 1.2: Map of the Ross Sea Region. Source: Topographic Map 135. Crown Copyright
Reserved. (http://www.linz.govt.nz/topography/topo-maps/antarctica) Inset showing key sites,
magnified in Figure 1.3. 1, Pennell Coast; 2, Ross Ice Shelf; 3, Amundsen Coast; 4, Siple Coast;
5, Shirase Coast; 6, Saunders Coast. ............................................................................................. 14
Figure 1.3: Magnified inset of the Ross Sea Region showing key sites. 1, Granite Harbour; 2,
Cape Bird; 3, Scott Base; 4, Minna Bluff; 5, Taylor Valley;........................................................ 15

Figure 2.1: Relative abundance of bacteria belonging to the phylum Actinobacteria and genus
Arthrobacter in soils of RSR determined by 16S rRNA gene clone libraries [5-7, 9, 23] and by
qPCR data (this study). ................................................................................................................. 59
Figure 2.2: Comparison of gene content in seven temperate Arthrobacter spp. and seven
Antarctic Arthrobacter isolates by COG categories. .................................................................... 62
Figure 2.3: Amino acid usage heatmap of seven temperate Arthrobacter spp. and seven
Antarctic Arthrobacter isolates based on their protein content. ................................................... 65
Figure 2.4: Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences from
seven Antarctic Arthrobacter isolates and seven temperate Arthrobacter species. ...................... 66
Figure 2.5: BLAST matrix of an all against all protein comparison of 14 Arthrobacter genomes.
....................................................................................................................................................... 67
Figure 2.6: MDS plots of BIOLOG substrate usage data of seven Antarctic Arthrobacter isolates
and three closely related temperate Arthrobacter species. a. Carbon utilisation profile determined
by BIOLOG PM1 and PM2A assay plates. b. Salinity tolerance profile determined by BIOLOG
PM9 assay plate. ........................................................................................................................... 77

Figure 3.1: Transmission electron micrograph of strain BrT ....................................................... 97
Figure 3.2: Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences from
strain BrT (highlighted in bold type) and closely related Paenibacillus species. ....................... 103

vi
Figure 4.1: Comparison of gene content in temperate Paenibacillus spp. and P. darwinianus
strains by COG categories. ......................................................................................................... 115
Figure 4.2: Amino acid usage heatmap of temperate Paenibacillus spp. and P. darwinianus
strains. ......................................................................................................................................... 117
Figure 4.3: A BLAST matrix of an all against all protein comparison of 12 Paenibacillus
genomes. ..................................................................................................................................... 118

vii
List of Tables
Table 1.1: Genomes of selected psychrophilic bacteria and archaea .......................................... 32

Table 2.1: General genome features of seven Antarctic Arthrobacter strains (this study) vs.
seven temperate Arthrobacter spp. [22]........................................................................................ 61
Table 2.2: List of genes linked to environmental stress response. ............................................... 70

Table 3.1: Distinctive phenotypic characteristics of strain BrT and closely related Paenibacillus
species ........................................................................................................................................... 99
Table 3.2: Cellular fatty acid compositions of strain BrT and closely related Paenibacillus
species ......................................................................................................................................... 101

Table 4.1: General genome features of P. darwinianus genomes (this study) vs. nine temperate,
soil-dwelling Paenibacillus spp. [43]. ........................................................................................ 114
Table 4.2: Oxidative and osmotic stress response associated genes in genomes of temperate
Paenibacillus spp. and P. darwinianus strains. .......................................................................... 122
Table 4.3: Cold-shock induced genes identified in genomes of temperate Paenibacillus spp. and
P. darwinianus strains. ................................................................................................................ 126
viii