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. 2013 Jun;7(6):1211-26.
doi: 10.1038/ismej.2013.8. Epub 2013 Feb 7.

Bacterial growth at -15 °C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1

Nadia C S Mykytczuk  1 , Simon J Foote, Chris R Omelon, Gordon Southam, Charles W Greer, Lyle G Whyte
Affiliations

Bacterial growth at -15 °C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1

Nadia C S Mykytczuk et al. ISME J. 2013 Jun.

Abstract

Planococcus halocryophilus strain Or1, isolated from high Arctic permafrost, grows and divides at -15 °C, the lowest temperature demonstrated to date, and is metabolically active at -25 °C in frozen permafrost microcosms. To understand how P. halocryophilus Or1 remains active under the subzero and osmotically dynamic conditions that characterize its native permafrost habitat, we investigated the genome, cell physiology and transcriptomes of growth at -15 °C and 18% NaCl compared with optimal (25 °C) temperatures. Subzero growth coincides with unusual cell envelope features of encrustations surrounding cells, while the cytoplasmic membrane is significantly remodeled favouring a higher ratio of saturated to branched fatty acids. Analyses of the 3.4 Mbp genome revealed that a suite of cold and osmotic-specific adaptive mechanisms are present as well as an amino acid distribution favouring increased flexibility of proteins. Genomic redundancy within 17% of the genome could enable P. halocryophilus Or1 to exploit isozyme exchange to maintain growth under stress, including multiple copies of osmolyte uptake genes (Opu and Pro genes). Isozyme exchange was observed between the transcriptome data sets, with selective upregulation of multi-copy genes involved in cell division, fatty acid synthesis, solute binding, oxidative stress response and transcriptional regulation. The combination of protein flexibility, resource efficiency, genomic plasticity and synergistic adaptation likely compensate against osmotic and cold stresses. These results suggest that non-spore forming P. halocryophilus Or1 is specifically suited for active growth in its Arctic permafrost habitat (ambient temp. ∼-16 °C), indicating that such cryoenvironments harbor a more active microbial ecosystem than previously thought.

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Figures

Figure 1
Figure 1
Arrhenius plot of log transformed growth rate constants for P. halocryophilus Or1 over the span of growth temperatures (37, 30, 25, 20, 15, 10, 5, 0, −5, −10, −15 °C). A characteristic biphasic growth trend is shown with the first phase (optimal) denoted in gray and the second phase (suboptimal) in black. Viable cell counts summarized to order of magnitude are shown on the secondary axis and correspond to maximum counts obtained in each growth rate experiment.
Figure 2
Figure 2
Mineralization assay of C-14 labeled acetate showing respiration by P. halocryophilus Or1 at subzero temperatures. Each point represents the mean cummulative mineralization (% 14CO2 recovered) from triplicate assays with s.e. shown. Curves are shown for 5 °C (▪), 0 °C (▴), −5 °C (□), −10 °C (*), −15 °C (●), −25 °C (♦) and the sterile controls represented by a single curve for the −5 °C control with a dotted line. All other control curves were similar to the one shown and were omitted from this figure for clarity. The line for −20 °C is also not shown as it overlapped with the −25 °C curve and was left out for clarity.
Figure 3
Figure 3
(a) Scanning electron microscope of cells grown at ambient temperatures, white arrows denote nodular feature being preserved along previous cell division planes. (b) Scanning electron microscope of cells grown at −15 °C in 18% NaCl and 7% glycerol, encrusted in dense nodular material. (c) Transmission electron microscopy of cross-section of dividing cells grown at −15 °C, noting the outer cell envelope with the nodular crust material in close association with the cell wall.
Figure 4
Figure 4
Functional distribution of genes within the P. halocryophilus Or1 genome classified by clusters of orthologous groups.
Figure 5
Figure 5
Genome-wide cold adaptation defined by five parameters that are used to determine cold adaptation at the protein level. The cold adaptation ratio (total cold adapted proteins/total hot adapted proteins) is shown to be significant above a threshold of 1.0 (right). The total number of proteins found to be significantly hot or cold adapted among 2038 of the P. halocryophilus Or1 genome coding sequence are shown per category (left).
Figure 6
Figure 6
Numbers of differentially expressed genes arranged by clusters of orthologous groups at −15 °C vs. control (25 °C), high salt vs. control (25 °C), and −15 °C vs. high salt. The data for upregulated and downregulated genes are shown above and below the line, respectively. clusters of orthologous group categories are the same as described in Figure 3.
Figure 7
Figure 7
Venn diagram illustrating the comparison between genes showing significant changes in expression among the three transcriptomic datasets for P. halocryophilus Or1 grown at control (25 °C), high salt (25 °C and 18% NaCl), and low temperature (−15 °C+18% NaCl) conditions. The number of genes found to be unique within and between treatments is shown denoting groups of genes that appear to be linked to either cold, salt, or common stress response.
Figure 8
Figure 8
Heat map of selected differentially expressed genes grouped by clusters of orthologous group category. Comparisons between transcriptomic data sets are shown in columns: (a) high salt vs −15 °C, (b) high salt vs 25 °C, (c) −15 °C vs 25 °C. Values are expressed as log2 transformed fold changes. All chosen genes were differentially expressed in at least one data set with P<0.05.
Figure 9
Figure 9
Summary of genome encoded adaptive traits (a), and the transcriptomic results for adaptive mechanism that were increased in expression and appear to be induced by high salt (b), or −15 °C (c). Traits that were similarly induced over basal levels under both stresses are outlined in black.
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