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

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Sequencing, assembly, annotation of the draft genome of Antarctic polyextremophile Nesterenkonia sp. AN1 and identification of putative molecular determinants underlying its polyextremophily

Introduction

The ice-free soils of terrestrial Antarctica constitute approximately 0.4 % of the continental land mass (Chan et al., 2013, Hopkins et al., 2006). These cold deserts represent the harshest dry terrestrial environments on Earth (Cary et al., 2010, Cowan et al., 2014). The dry soils are characterized by extreme cold, with frequent wide temperature fluctuations (Aislabie et al., 2006, Dreesens et al., 2014), low water availability, high alkalinity and salinity, elevated levels of UV irradiation and scarcity of nutrients (Aislabie et al., 2006, De Maayer et al., 2014). Despite the harsh physico-geochemical conditions, these soils harbour abundant microbial communities, particularly within refugia where the organisms are shielded from direct impact of some elements of the harsh conditions (Cowan et al., 2014).
On the basis of fitness, microorganisms living in Antarctica are categorized as either specialists (e.g. obligate psychrophiles) or generalists (e.g. psychrotolerant microorganisms) (Vincent, 2000). The former are adapted to survive optimally under the Antarctic ambient environmental conditions. The generalists are further classified into two types, those surviving sub-optimally (Type I) and those capable of adjusting between optimal and suboptimal growth (Type II) depending on the prevailing environmental conditions (Vincent, 2000). Several bacterial isolates from the Antarctic terrestrial habitats exhibit psychrotrophic adaptations. Examples include strains of Arthrobacter and Paenibacillus isolated from soils in the Ross Sea region (Dsouza et al., 2014, Dsouza et al., 2015). The predominance of psychrotolerant bacteria in Antarctic dry soils has been attributed to the evolution of essential adaptive strategies which enable the organisms to cope with frequent and wide temperature fluctuations in the Dry Valleys (Kirby et al., 2011). Very little is known regarding the precise mechanisms that determine survival of these microorganisms (Chan et al., 2013). However, functional and community ecology of the Antarctic dry soils have shown that Actinobacteria are among the most successful colonizers of these cold arid soils (Geyer et al., 2014, Schwartz et al., 2014).
The Gram-positive genus Nesterenkonia belongs to the family Micrococcaceae (Stackebrandt, 2012, Stackebrandt et al., 1995) and can be easily distinguished from other members of the family by the morphological features, G +C content, isoprenoid quinones and composition of peptidoglycans of Nesterenkonia spp. (Goodfellow, 2012). Nesterenkonia spp. are generally aerobic, catalase positive, chemo-organotrophic and haloalkaliphilic (Collins et al., 2002, Li et al., 2005, Stackebrandt et al., 1995). They are usually coccoid or rod-shaped, with or without branching (Stackebrandt et al., 1995). They are non-spore forming, and non-encapsulated and the genomic DNA is characterized by a high G+C content of 64 % to 72 % (Li et al., 2005). The isoprenoid quinones are mainly comprised of menaquinones with seven, eight and nine isoprene units which are completely unsaturated. The peptidoglycans in Nesterenkonia are of the A4α type (Stackebrandt, 2012, Stackebrandt et al., 1995).
Stackebrandt and co workers proposed the emendation of the genus Micrococcus after a detailed phylogenetic and chemotaxonomic evaluation of its members (Stackebrandt et al., 1995). Consequently, Micrococcus halobius was reclassified and named Nesterenkonia halobia. Further affiliation of new strains to the genus were recommended to be on the basis of mena-quinone composition, types of peptidoglycans, morphology and 16S ribosomal RNA (Stackebrandt, 2012). The genus Nesterenkonia is currently comprised of thirteen validly described species (Parte, 2013).
Members of the genus Nesterenkonia are isolated from a wide range of environmental sources. These include cotton and paper mills in China (Luo et al., 2008, Luo et al., 2009), fermented seafood in Korea (Yoon et al., 2006) as well as faeces of an AIDS patient in France (Edouard et al., 2014). Predominantly, however, Nesterenkonia spp. are isolated from extreme environments. Several strains have been reported mainly from saline environments, such as desert and saline soils in Egypt and China, respectively (Li et al., 2005, Li et al., 2004, Li et al., 2008), hyper-saline lake in east Antarctica (Collins et al., 2002) and a soda lake in Ethiopia (Delgado et al., 2006).
Nesterenkonia sp. AN1 was isolated from soil samples collected from the Miers Valley, which forms part of the McMurdo Dry Valleys of Antarctica (Nel et al., 2011). Nesterenkonia sp. AN1 represents the first reported psychrophilic member of the genus. Like other species in the genus, Nesterenkonia sp. AN1 is an obligate alkaliphile, with pH requirement of 9 – 10 and halotolerant, capable of optimal growth at 0 to 15 % (wt/vol) salt concentration (Li et al., 2005, Nel et al., 2011). As for several other bacterial isolates from the cold desert soils, Nesterenkonia sp. AN1 survives temperatures well below the optimum growth temperature of psychrophiles (15°C) but has an optimal growth temperature of 21°C, and is hence classified as ‘psychrotolerant’ (Nel et al., 2011). Furthermore, a novel aliphatic amidase (superfamily nitrilase) that is active on short chain amides has been isolated and characterized in Nesterenkonia sp. AN1 (Nel et al., 2011).
Here we present the high-quality draft genome sequence of Nesterenkonia sp. AN1 and highlight the important adaptive features identified from the genome sequence, which are likely to underscore its polyextremophily.

Material and Methods

Genome Sequence of Nesterenkonia sp. AN1

Culturing and genomic DNA extraction

Nesterenkonia sp. AN1 was routinely cultured at 21°C in modified Castenholz media (Nel et al., 2011). Cultures were streaked on modified Castenholz agar plates and maintained at 21°C. The colonies were scraped from the plates and transferred to three 50ml sterile tube containing Castenholz broth.
Genomic DNA was extracted using a modified bead beating phenol/chloroform extraction protocol (http://www.utoledo.edu/search.html?q=DNA extraction soil.pdf). Briefly, pooled colonies for the replicated cultures were centrifuged for 5 min at 14000 rpm to obtain strong pellets. The pellets were re-suspended in 1 ml of extraction buffer (50 mMNaCl, 50 mMTris-HCl, 50 mM EDTA and 5 % SDS; pH 8). Each suspension was transferred to a sterile 2 ml safe-locks tube containing 0.4 ml of 0.10 mm glass beads. The cells were homogenized using the PowerLyserTM 24 Bench Top Beed-Based Homogenizer at 2,000 rpm for five minutes as per the manufacturer’s recommendations. The supernatant was transferred to a new 2ml eppendorf tube and 300 µl of phenol and chloroform/isoamyl alcohol were added. The preparation was mixed by vortexing for 10 seconds and centrifuged at 14000 rpm. The DNA contained in the upper aqueous phase was into a new 2 ml eppendorf tube. Further extraction of DNA was completed using 500 µl of chloroform only. The aqueous upper phase was collected into a 1.5 ml micro centrifuge tube. To improve the purity of the genomic DNA, an RNA removal step was incorporated by adding 1 µl of RNase A to the sample. The sample was incubated at 37°C for 60 minutes. The genomic DNA was precipitated by adding 0.1 and 0.7 volumes of 3 M sodium acetate and isopropanol, respectively and centrifuged at 14,000 rpm for 30 min at 10°C. The supernatant was aspirated carefully. The pellet was washed using 0.5 ml 70 % ice-cold ethanol and centrifuged at maximum speed for 5 min to re-pellet the DNA.
The pellet then re-suspended in 50 µl DNase/RNase-free water. The quality and quantity of the extracted DNA was assessed using a NanodropTM spectrometer, Qubit ® 2.0 fluorimeter and visualized by electrophoresis on a 1 % agarose gel.

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Genome Sequencing and assembly

A first sequencing run was done using Illumina GA IIx chemistry at the University of Western Cape. Because of the challenges associated with assembling the GA IIx short reads, a second sequencing run was conducted using the Ion torrent PGM at the University of Pretoria sequencing facility. The quality of the reads was assessed using the sequencing QC report tool in CLC Genomics Workbench v. 6 (CLC BIO: http://www.clcbio.com/products/clc-genomics-workbench). The reads were assembled de novo using several commercial and publically available assemblers, including Velvet v1.2.10 (Zerbino and Birney, 2008), CLC Genomics Workbench v. 6 (CLC BIO: http://www.clcbio.com/products/clc-genomics-workbench) and the Seqman NGen assembler v11 (DNAStar – http://www.dnastar.com/t-nextgen-seqman-ngen.aspx). To generate highly accurate genome assemblies, the assembled contiguous sequences (contigs) from the individual assemblers were compared using Mauve (Darling et al., 2010) and Bioedit v 7.2.5 (Hall, 1999, Hall, 2011). The contigs from CLC and NGen were assembled further into longer contigs (‘scaffolds’) using different in silico strategies including local BLAST search within and between contigs from different assemblers using Bioedit v 7.2.5 (Hall, 1999, Hall, 2011) and searching for open reading frames using NCBI-ORF finder (Sayers et al., 2011). Final decision to extend contigs was based on the comparison of the Nesterenkonia sp. AN1 contigs against the draft genomes sequences of Nesterenkonia sp. F, Nesterenkonia sp. NP1 and N. alba DSM 19423, obtained from the NCBI database using Mauve (Darling et al., 2004, Darling et al., 2010).

Genome Annotation

The draft genome of Nesterenkonia sp. AN1 was uploaded into several structural annotation pipelines, including the Rapid Annotation using Subsystem Technology (RAST) server (Overbeek et al., 2014), FGENESB (Solovyev and Salamov, 2011), GeneMark.hmm (prokaryotic version 2.8) (Borodovsky and Lomsadze, 2011, Borodovsky and Lomsadze, 2013), the NCBI prokaryotic genome annotation pipeline (Tatusova et al., 2013) and the Bacterial Annotation SYStem (BASys) pipeline (Van Domselaar et al., 2005). Protein coding sequences (CDSs) that were identified as hypothetical proteins were assessed using the NCBI conserved domain batch search tool (Marchler-Bauer et al., 2015). To assign putative functions to the CDSs, the proteins were functionally annotated using eggNOG non-supervised orthologous groups (Powell et al., 2013). The genes encoding tRNA were predicted using the tRNA prediction program ARAGORN v. 1.2 (Laslett and Canback, 2004). Cellular localization for all the predicted proteins of Nesterenkonia sp. AN1 was determined using CELLO2GO (Yu et al., 2014). The tool uses a combination of CELLO (Yu et al., 2006) localization methods and Blast analysis of characterised proteins with GO annotation. CELLO2GO has been reported to predict subcellular localization in Gram-positive bacteria with 99.4 % accuracy (Yu et al., 2014).
The draft genome sequence was deposited and made public on the NCBI under the GenBank accession number NZ_JEMO00000000 (GI: 738529954) and NCBI Reference Sequence number NZ_JEMO00000000.1. The draft genome is also available via the genomes online database (GOLD) under the accession number Gp0085412.

Identification of genomic islands, prophages and extrachromosomal elements

The draft genome of Nesterenkonia sp. AN1 was queried for the presence of genomic islands (GIs) using IslandViewer 3 (Dhillon et al., 2015). The predicted features in the various GIs were further assessed via BlastP searches against the NCBI RefSeq and non-redundant (nr) protein databases (Jenuth, 2000). The genome was also analysed for the presence of phage elements using the online PHAST tool (Zhou et al., 2011).

Identification of stress response mechanisms

The proteins encoded on the genome of Nesterenkonia sp. AN1 were screened to identify proteins with putative adaptive functions which have been reported in the literature (Dsouza et al., 2015, Medigue et al., 2005). Protein functions were also determined on the basis of the Nesterenkonia sp. AN1 annotations obtained using RAST subsystems (Overbeek et al., 2014), BASys bacterial annotation pipeline (Van Domselaar et al., 2005), eggNOG classification (Powell et al., 2013, Powell et al., 2012) and via BLASTP analysis using the NCBI RefSeq and non-redundant (nr) protein databases (Jenuth, 2000).

Results and Discussion

Genome sequencing and assembly

The first sequencing run, using the Illumina GA IIx, yielded 5,177,635 reads of 44 bp average length, with an estimated genome coverage of ~ 36x. The assembly of these reads produced a large number of contigs, probably due to the presence of repeat regions in the genome. This prompted additional sequencing using the Ion Torrent chemistry, which yields longer reads. The Ion Torrent PGM yielded 3,842,066 reads of mean length and coverage of 324 bp and ~ 351x, respectively.
The reads from both Illumina GA IIx and Ion-torrent PGM platforms were assembled de novo using CLC Genomics Workbench v. 6, DNAStar Seqman NGen v11 and Velvet v1.2.10 (Table 2-1). By applying different in silico techniques, contigs derived from the Illumina GAIIx reads were assembled into 410 contigs of ~ 2.98 megabases (Mb), with a GC content of 67.48 % and a contig length range of between 133 and 62,284 Mb. Regions of overlap between these contigs and those produced from Ion-torrent PGM reads were identified using Mauve (Darling et al., 2004, Darling et al., 2010) and the information was utilised to improve the assembly of the Ion-torrent PGM reads. The final genome assembly from the Ion-torrent PGM yielded a genome of ~ 3.04 Mb assembled in 41 contigs ranging between 1,439 and 339,148 nucleotides in length with a mean G+C content 67.42 %.

Declaration 
Acknowledgement 
Abstract
Table of contents 
List of figures .
List of tables 
List of appendices
Chapter 1  Extremophiles in the ‘omics’ era 
1.1 Introduction
1.2 Extremophiles
1.2.1 Psychrophilic (Cold-adapted) microorganisms
1.2.2 Halophilic (Salt Adapted) Microorganisms
1.2.3 Alkaliphiles (High pH-Adapted) Microorganisms
1.2.4 Industrial applications of extremophiles
1.3 Genomics and ‘omic technologies
1.3.1 Genomics
1.3.1.1 Sequencing technologies
1.3.1.2 Genome assembly
1.3.1.3 Genome annotation
1.3.2 Comparative genomics
1.3.3 ‘Omic’ technologies
1.3.3.1 Transcriptomics
1.3.3.2 Proteomics and other ‘omics technologies
1.4 Extremophiles in the ‘omics’ era
1.4.1 Psychrophiles in the ‘omics’ era
1.4.2 Halophiles in the ‘omics’ era
1.4.3 Alkaliphiles in the ‘omics’ era
1.5 Conclusions
1.6 References
Chapter 2 Sequencing, assembly, annotation of the draft genome of Antarctic polyextremophile
Nesterenkonia sp. AN1 and identification of putative molecular determinants underlying its polyextremophily 
2.1 Introduction
2.2 Material and Methods
2.3 Genome Sequence of Nesterenkonia sp. AN1
2.4 Results and Discussion
2.5 Stress response mechanisms
2.6 Conclusion
2.7 References
Chapter  3 Comparative genomic analyses of four Nesterenkonia species 
3.1 Introduction
3.2 Materials and Methods
3.3 Results and Discussion
3.4 Conclusion
3.5 References
Chapter 4 Transcriptome analysis reveals several key adaptive features for the survival of Nesterenkonia sp. AN1 under cold conditions 
4.1 Introduction
4.2 Material and Methods
4.3 Results and Discussion
4.4 Conclusions
4.5 References
Chapter 5 General conclusions
5.1 Introduction
5.2 Adaptations of the Antarctic Nesterenkonia
5.3 Concluding remarks and future perspectives
5.4 References
Appendices
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