ABSTRACT: Permafrost soils are extreme environments that exert low-temperature, desiccation and starvation stress on bacteria over thousands to millions of years. To understand how Psychrobacter arcticus 273-4 survived for > 20,000 years in permafrost, transcriptome analysis was performed during growth at 22°C, 17°C, 0°C, and -6°C using a mixed effects ANOVA model. Genes for transcription, translation, energy production and most biosynthetic pathways were down-regulated at low temperatures. Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943) and energy efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by up-regulation at low temperature including genes for the biosynthesis of proline, tryptophan, methionine, and histidine. RNases and peptidases were generally up-regulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of the stringent response by relA activity. In contrast to results observed in other psychrophiles and mesophiles, only clpB and hsp33 were up-regulated at low temperature with no up-regulation of other chaperones and peptidyl-prolyl isomerases. Knockout mutants of relA, csdA, and dac2 were all deficient in low temperature growth, but a mutant in dac1 was deficient in growth at 17°C. The combined data suggest that the basal biological machinery including translation, transcription and energy metabolism are well adapted to function across the -6°C to 22°C growth range of P. arcticus and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth. Cy-dye labelled cDNA populations were generated from total RNA extracted from mid-exponential growth phase cultures of Psychrobacter arcticus 273-4 grown at 22°C, 17°C (optimal growth rate), 0°C, and -6°C. All pairwise temperature comparisons were performed in this 4-level single factor experiment with dye swapping nested within biological replicate. Five biological replicates (20 samples totalled over all temperatures) were included in each comparison for a total of 30 hybridizations. Data were analyzed using R/MAANOVA with an ANOVA model which included gene, dye, biological replicate, slide, and growth temperature terms (with interactions). Spike-in mRNAs were included immediate following the cell lysis stage in order to serve as monitors for RNA degradation from lysis through hybridization.
