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 Or1that 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 proteinswas 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 Biol5:R12.
Pearson MD, Noller HF. (2011). The draft genome of Planococcus donghaensis MPA1U2 reveals
nonsporulation pathways controlled by a conserved Spo0A regulon. J Bacteriol193(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 expressedas 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 / Best hit score / Genome Size (Mb)Bacillus B-14905 / 502 / 4.5
Lysinibacillus sphaericus C3-41 / 383 / 4.6
Bacillus licheniformis ATCC 14580 / 370 / 4.2
Geobacillus sp. WCH70 / 307 / 3.5
Anoxybacillus flavithermus WK1 / 300 / 2.8
Planococcus donghaensis MPA1U2 / 250 / 3.3
Exiguobacterium sibiricum 255-15 / 160 / 3.0
Colwellia psychrerythraea 34H / 7 / 5.3
Psychrobacter arcticus 273-4 / 4 / 2.6
Table S2. Results for the amino acid distribution and adaptive index analyses for significantly hot or cold adapted genes
Cold adapted / Hot adapted / TotalHydrophobicity / 356 / 540 / 896
Proline / 797 / 241 / 1038
Aliphacity / 448 / 431 / 879
Acidic Residues / 708 / 352 / 1060
Arg/Lys / 294 / 385 / 679
Total / 2603 / 1949
Total CDS analyzed / 2463
Total genes with swiss prot hits / 2038
Hypothetical genes excluded / 1404
RNAs excluded / 65
Table S3. Genomic comparisons ofosmoregulatory genes for choline/glycine betaine uptake and synthesis. Genomic redundancy is built in to P. halocryophilus Or1 genome with multiple glycine betaine uptake genes.
OrganismProP / ProV / ProW / ProX / OpuD / OpuAA / OpuAB / OpuAC / BetB / BetT / Glyc
Planococcus halocryophilus Or1 / X / X(3) / X / X(2) / X(5) / X(2) / X(2) / X(2) / X / X
Psychrobacter cryohalolentis K5 / X / X / X / X
Psychrobacter arcticus 273-4 / X / X
Psychromonas ingrahamii / X(3) / X(3) / X / X
Colwellia psychrerythraea 34H / X / X / X / X(3)
Desulfotalea psychrophila / X / X
Exiguobacterium sibiricum 255-15 / X / X / X / X / X / X / X
X, presence of gene; (#), number of copies in genome
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) / 60.3 / 20.8 / 42.7
Total filtered reads / 597654 / 206235 / 422805
Read length (bp) / 101 / 101 / 101
G+C% / 40 / 45 / 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.