Genomics of Antarctic polyextremophile Nesterenkonia sp. AN1

241 Pages·2016·7.76 MB·English

The Antarctic polyextremophilic Nesterenkonia

Genomics of Antarctic polyextremophile Nesterenkonia

By

Habibu Aliyu

Submitted in partial fulfilment of the requirements for the degree

Philosophiae Doctor (PhD) Genetics

In the Faculty of Natural & Agricultural Sciences

University of Pretoria

Pretoria

April 2016

Supervisor: Professor Don A Cowan

Co-supervisor: Dr Pieter de Maayer

© University of Pretoria
The Antarctic polyextremophilic Nesterenkonia
“Who taught by the pen-

Taught man that which he knew not.”

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© University of Pretoria
The Antarctic polyextremophilic Nesterenkonia
Declaration

I, Habibu Aliyu, declare that the thesis, which I hereby submit for the degree of Philosophiae

Doctor (PhD) in Genetics at the University of Pretoria, is my own work and has not been

previously been submitted by me for a degree at this or any other tertiary institution.

SIGNATURE: ..…………………………..

DATE: ………………………………….

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The Antarctic polyextremophilic Nesterenkonia
Acknowledgement

I would like to extend my sincere gratitude to my supervisor Professor Don A Cowan, who

provided me the rare and enviable opportunity to conduct my PhD research at the Centre for

Microbial Ecology and Genomics (CMEG). Prof, I appreciate your words of wisdom, immense

knowledge, and sense of humour and above all, the unequalled academic leadership and

mentoring style.

My sincere appreciation goes to my co-supervisor Dr Pieter de Maayer. Pieter, you patiently

and firmly taught me even when at times I was stubbornly unwilling to shift ground. I greatly

appreciate your ingenuity, patience, insightful comments, the encouragements and your

resourcefulness.

I want to thank everyone at CMEG, in particular, I am grateful to Enrico, Leandro and Surendra

for the bioinformatics support.

I would like to thank Dr Abdullahi A Yusuf and his family for their kindness and support

throughout my stay in Pretoria. My sincere appreciation goes to Alhaji Mustafa Atiku (thank

you sir for everything), Mal. Abdullahi Magauri, Professors AK Adamu, ML Balarabe and

Kabiru Bala, those calls have uplifted my spirit and often times they come when I needed them

most. I thank Abubakar Khalid, Hakeem, Nangamso and Usman Danbaba, for their kindness

and friendliness.
Last but not the least, I would like to extend my sincere gratitude to my wife Suwaiba and our

beloved kids, Aliyu and Aisha for their unconditional love, support, prayers and understanding.

My thanks and love to my parents, Rabiatu and Aliyu M for everything. Many thanks to my

siblings, Dr Muhammad, Khalid, Abdullahi, Fatima, Aisha, Ibrahim, Idris and Rahma, thank

you all for your kindness and love.

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© University of Pretoria
The Antarctic polyextremophilic Nesterenkonia
Abstract

Nesterenkonia sp. AN1 was isolated from soil samples obtained from the Miers Valley,

McMurdo region in eastern Antarctica, where the organism survives under 'extreme'

environmental conditions, including extreme cold, osmotic and oxidative stresses, desiccation

and starvation as well as elevated fluxes of ultraviolet radiation. The isolate is the first member

of the genus Nesterenkonia to be reported from Antarctic soils. This thesis provides insights

into the molecular determinants underlying the ability of the Antarctic Nesterenkonia sp. AN1

to survive under multiple stress conditions.

A range of genomic and comparative genomic analyses was applied to elucidate the genetic

determinants underlying survival strategies of Nesterenkonia sp. AN1. To highlight

psychrotolerance mechanisms, the transcriptome of the Nesterenkonia sp. AN1grown at 5 ºC

and 21 ºC were also compared.

The draft genome of Nesterenkonia sp. AN1 represents the first genome sequence of a cold

adapted member of the genus Nesterenkonia. The genome was 3.04 Mb in size, was assembled

into forty-one contigs and encoded 2,852 proteins. These proteins include those putatively

involved in cold shock, cold acclimation, osmotic and oxidative stress responses as well as the

modulation of membrane fluidity.

Genome-wide comparisons of th e cold-active Nesterenkonia sp. AN1 and three mesophilic
strains revealed that members of the genus Nesterenkonia encodes an open pan-genome, which

is consistent with their ecological diversity and niche specializations. Further evaluation of the

pan-genome compartments showed that the majority of the adaptation genes are shared by all

the strains, suggesting that members of the genus are naturally resilient to environmental

stresses. The genome of Nesterenkonia sp. AN1, however, encodes unique putative adaptation

proteins within the strain-specific genome fraction and also showed evidence of molecular

evolution in a large number of the core genes. These events indicate that the evolution of the

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The Antarctic polyextremophilic Nesterenkonia
Nesterenkonia sp. AN1 genome may have involved a significant number of gene acquisition

events and the incorporation of advantageous mutations in the ancestral genetic stock.

Transcriptome data of Nesterenkonia sp. AN1 revealed that ~ 97 % of the genes predicted in

Nesterenkonia sp. AN1 were expressed under the experimental conditions. Analyses of the

transcriptome showed that there was significant induction at 5 ºC of transcripts that coded for

proteins associated with antioxidant activity. There was also overexpression of universal stress

protein genes related to uspA, along with genes encoding other characterised cold stress

features. Genes encoding key enzymes of the glyoxylate cycle were also induced at 5 ºC,

suggesting possible adaptation strategies for energy metabolism in cold habitats.

Taken together, the combined data revealed the presence of pervasive adaptive strategies which

likely determine niche specialization of Nesterenkonia sp. AN1 and support its survival in the

arid soils of Antarctica.

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The Antarctic polyextremophilic Nesterenkonia
Table of contents

Declaration ............................................................................................................................... III

Acknowledgement ................................................................................................................... IV

Abstract ..................................................................................................................................... V

Table of contents .................................................................................................................... VII

List of figures ......................................................................................................................... XII

List of tables ........................................................................................................................... XV

List of appendices ................................................................................................................. XVI

Chapter 1 ................................................................................................................................... 1

1 Extremophiles in the ‘omics’ era ........................................................................................ 2

1.1 Introduction ................................................................................................................................ 2

1.2 Extremophiles ............................................................................................................................ 3

1.2.1 Psychrophilic (Cold-adapted) microorganisms ................................................................. 5

1.2.2 Halophilic (Salt Adapted) Microorganisms ....................................................................... 8

1.2.3 Alkaliphiles (High pH-Adapted) Microorganisms .......................................................... 10

1.2.4 Industrial applications of extremophiles ......................................................................... 11

1.3 Genomics and ‘omic technologies ........................................................................................... 12

1.3.1 Genomics......................................................................................................................... 12

1.3.1.1 Sequencing technologies .............................................................................. 14

1.3.1.2 Genome assembly ........................................................................................ 16

1.3.1.3 Genome annotation ...................................................................................... 18

1.3.2 Comparative genomics .................................................................................................... 19

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The Antarctic polyextremophilic Nesterenkonia
1.3.3 ‘Omic’ technologies ........................................................................................................ 22

1.3.3.1 Transcriptomics ............................................................................................ 22

1.3.3.2 Proteomics and other ‘omics technologies .................................................. 24

1.4 Extremophiles in the ‘omics’ era ............................................................................................. 25

1.4.1 Psychrophiles in the ‘omics’ era ...................................................................................... 26

1.4.2 Halophiles in the ‘omics’ era ........................................................................................... 29

1.4.3 Alkaliphiles in the ‘omics’ era......................................................................................... 31

1.5 Conclusions .............................................................................................................................. 32

1.6 References ................................................................................................................................ 33

Chapter 2 ................................................................................................................................. 50

2 Sequencing, assembly, annotation of the draft genome of Antarctic polyextremophile

Nesterenkonia sp. AN1 and identification of putative molecular determinants underlying its

polyextremophily ..................................................................................................................... 51

2.1 Introduction .............................................................................................................................. 51

2.2 Material and Methods .............................................................................................................. 54

2.3 Genome Sequence of Nesterenkonia sp. AN1 ......................................................................... 54

2.3.1 Culturing and genomic DNA extraction .......................................................................... 54

2.3.2 Genome Sequencing and assembly ................................................................................. 55

2.3.3 Genome Annotation ........................................................................................................ 56

2.3.4 Identification of genomic islands, prophages and extrachromosomal elements ............. 57

2.3.5 Identification of stress response mechanisms ................................................................. 57

2.4 Results and Discussion ............................................................................................................ 58

2.4.1 Genome sequencing and assembly .................................................................................. 58

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2.4.2 Genome annotation ......................................................................................................... 59

2.4.3 Genomic islands, prophages and other extrachromosomal elements .............................. 63

2.5 Stress response mechanisms .................................................................................................... 67

2.5.1 Cold stress response ........................................................................................................ 68

2.5.1.1 Cold shock proteins and cold acclimation proteins ..................................... 68

2.5.1.2 Membrane adaptations ................................................................................. 70

2.5.2 Oxidative stress response ................................................................................................ 71

2.5.3 Protection against osmotic stress..................................................................................... 72

2.5.4 General stress response ................................................................................................... 73

2.6 Conclusion ............................................................................................................................... 74

2.7 References ................................................................................................................................ 75

Chapter 3 ................................................................................................................................. 85

3 Comparative genomic analyses of four Nesterenkonia species ........................................ 86

3.1 Introduction .............................................................................................................................. 86

3.2 Materials and Methods ............................................................................................................. 88

3.2.1 Genome sequences and structural annotation ................................................................. 88

3.2.2 Phylo-genomic analyses .................................................................................................. 88

3.2.3 “Pan-genome” analysis ................................................................................................... 89

3.2.4 Detection of positive selection ........................................................................................ 90

3.2.4.1 Determination of Orthologous gene sets...................................................... 90

3.2.4.2 Detection of recombination and positive selection ...................................... 90

3.3 Results and Discussion ............................................................................................................ 92

3.3.1 General genome metrics .................................................................................................. 92

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3.3.2 Phylo-genomics of Nesterenkonia spp ............................................................................ 96

3.3.3 The ‘pan-genome’ of four Nesterenkonia strains .......................................................... 100

3.3.3.1 Comparative genomics identifies unique stress response proteins in

Nesterenkonia sp. AN1 .............................................................................................. 105

3.3.4 Signatures of positive selection are prevalent in the core genome of Nesterenkonia ... 107

3.3.4.1 Orthologs sets and alignment strategy ....................................................... 107

3.3.4.2 Recombination and positive selection ....................................................... 108

3.4 Conclusion ............................................................................................................................. 115

3.5 References .............................................................................................................................. 117

Chapter 4 ............................................................................................................................... 126

4 Transcriptome analysis reveals several key adaptive features for the survival of

Nesterenkonia sp. AN1 under cold conditions ....................................................................... 127

4.1 Introduction ............................................................................................................................ 127

4.2 Material and Methods ............................................................................................................ 128

4.2.1 Growth conditions ......................................................................................................... 128

4.2.2 RNA preparation ........................................................................................................... 129

4.2.3 RNA sequencing ............................................................................................................ 130

4.2.4 RNA-Seq analysis ......................................................................................................... 130

4.3 Results and Discussion .......................................................................................................... 131

4.3.1 RNA-Seq read mappings ............................................................................................... 131

4.3.2 Analysis of gene expression .......................................................................................... 132

4.3.2.1 Most stress response genes were expressed under both cold and optimal

growth conditions....................................................................................................... 135

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