Supplementary material
Methods:
Additional genome assembly information
Some gap closures were facilitated by comparison against the P. donghaensis MPA1U2 genome
(Accession AEPB00000000; Pearson and Noller, 2011) using MUMmer 3.22 (Kurtz et al., 2004).
Insertion sequences/transposase searches were performed using IS Finder (Siguier et al., 2006). Of the
35 contigs, 24 of them included rRNA or transposase sequences on their 5' or 3' ends, but further
manual gap closures were not completed.
Genome-wide cold adaptation ratio
The cold adaptation ratio based on amino acid usage was determined for each of the 2038
proteins from P. halocryophilus Or1 that had homologous hits within the Swiss-Prot database (i.e.,
non-hypothetical) were compared against the five closest (mesophilic) matches. Values for each
parameter were calculated using in-house PERL scripts and the difference between the P.
halocryophilus Or1 protein and the average of the five mesophilic proteins was compared using a
student t-test (p = 0.05). Each of the 2038 proteins was then classified as significantly cold or hot
adapted for a given parameter and the sum of cold adapted proteins vs. total hot adapted was used to
define the cold adaptation ratio for P. halocryophilus Or1 genes.
References:
Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. (2004).
Versatile and open software for comparing large genomes. Genome Biol 5:R12.
Pearson MD, Noller HF. (2011). The draft genome of Planococcus donghaensis MPA1U2 reveals
nonsporulation pathways controlled by a conserved Spo0A regulon. J Bacteriol 193(21):6106.
Siguier P, Filee J, Chandler M. (2006). Insertion sequences in prokaryotic genomes. Curr Opin
Microbiol 9:526–531.
Figure S1. Viable cell counts determined with LiveDead (Invitrogen) staining and fluorescent imaging.
Images show live (a) and dead (b) cells from cultures grown at 23°C (top) and -15°C (bottom).
Figure S2. Heat map of selected differentially expressed genes classified as conserved and hypothetical
protein COG categories. Comparisons between transcriptomic datasets are shown in columns: (a.) high
salt vs. -15°C, (b.) high salt vs. 25°C, (c.) 25°C vs -15°C. Values are expressed as log2 transformed
fold changes. All chosen genes were differentially expressed in at least one dataset with P < 0.05.
Table S1. Closest neighbours to the P. halocryophilus Or1 genome based on similarity of homologous
protein sequences (> 70% identity), along with genome comparisons against known cold tolerant
organisms.
Organism ID
Bacillus B-14905
Lysinibacillus sphaericus C3-41
Bacillus licheniformis ATCC 14580
Geobacillus sp. WCH70
Anoxybacillus flavithermus WK1
Planococcus donghaensis MPA1U2
Exiguobacterium sibiricum 255-15
Colwellia psychrerythraea 34H
Psychrobacter arcticus 273-4
Best hit score
502
383
370
307
300
250
160
7
4
Genome Size (Mb)
4.5
4.6
4.2
3.5
2.8
3.3
3.0
5.3
2.6
Table S2. Results for the amino acid distribution and adaptive index analyses for significantly hot or
cold adapted genes
Hydrophobicity
Proline
Aliphacity
Acidic Residues
Arg/Lys
Total
Total CDS
analyzed
Total genes with
swiss prot hits
Hypothetical
genes excluded
RNAs excluded
Cold adapted
356
797
448
708
294
2603
Hot adapted
540
241
431
352
385
1949
Total
896
1038
879
1060
679
2463
2038
1404
65
Table S3. Genomic comparisons of osmoregulatory genes for choline/glycine betaine uptake and
synthesis. Genomic redundancy is built in to P. halocryophilus Or1 genome with multiple glycine
betaine uptake genes.
Organism
ProP ProV ProW ProX OpuD OpuAA OpuAB OpuAC BetB BetT Glyc
X X(3) X X(2) X(5) X(2)
Planococcus
halocryophilus
Or1
X
X
X
Psychrobacter
cryohalolentis
K5
X
Psychrobacter
arcticus 273-4
X(3) X(3)
Psychromonas
ingrahamii
X
X
X
Colwellia
psychrerythraea
34H
X
X
Desulfotalea
psychrophila
X
X
X
Exiguobacterium X
sibiricum 255-15
X, presence of gene; (#), number of copies in genome
X(2)
X(2)
X
X
X
X
X
X
X(3)
X
X
X
Table S4. Summary statistics for Illumina sequenced transcriptomic datasets derived from cultures
grown at low temperature and high salt conditions (-15°C) and high salt conditions (18% salt) versus
control conditions (25°C)
Total sequence
information (Mbp)
Total filtered reads
Read length (bp)
G+C%
Control (25°C)
60.3
Cold stress (-15°C)
20.8
High salt (18%)
42.7
597654
206235
422805
101
40
101
45
101
41
Contaminating rRNA* None
None
None
* All samples were treated for mRNA enrichment (Microbe Express) and no residual rRNA
contaminant sequences were found in the transcriptome libraries.