# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion
### Changes in Amino Acid Metabolism and Translation-Related Pathways in Response to Cold Stress  
After 90 min of cold stress, we observed a significant downregulation of metabolic pathways of several amino acids in _S. baltica_, including arginine, valine, leucine and isoleucine, phenylalanine, tyrosine, tryptophan, arginine, proline, histidine, phenylalanine, and beta-alanine metabolism. Downregulation of these pathways was still present at 180 min; moreover, the downregulated pathways also included glycine, serine, threonine, lysine and tyrosine metabolism (Supplementary Figure S1 and Table S3). Most of these pathways were characterized with downregulation of 50% or more genes assigned to each pathway; valine, leucine and isoleucine metabolism appeared to be the most affected pathway with over 80% of assigned genes being significantly downregulated (Supplementary Table S3). Interestingly, initially mostly non-polar amino acid metabolism was transcriptionally downregulated, but later polar amino acid metabolism was affected as well. It was previously shown that _S. oneidensis_  
Figure 6: Expression changes in _S. baltica_ fatty acid metabolism upon cold stress. Heatmap showing row _Z_-score scaled normalized expression for corresponding genes. Column 0 stands for control sample; replicates for 90 and 180 min time points are labeled R\({}_{1}\) and R\({}_{2}\). downregulates genes of amino acid biosynthesis under cold stress conditions [23]. A study of cold stress-induced proteomic response confirmed that a variety of _S. oneidensis_ proteins used in amino acid synthesis was also downregulated [93]. Results of functional annotation of the differentially expressed genes from _S. piezotolerans_ showed upregulation of amino acid transport and metabolism-related genes upon heat stress, and downregulation upon cold stress [94]. Contrary, analysis of cold stress response in _T. tengcongensis_ revealed that among differentially expressed genes, those involved in synthesis of branched amino acids were upregulated [55]. Interestingly, along with downregulation of amino acid metabolism, we observed upregulation of some translation-related pathways. Our results of functional annotation of differentially expressed genes from _S. baltica_ showed upregulation of the aminoacyl-tRNA biosynthesis pathway (Supplementary Figure S1); also, expression of a set of aminoacyl synthetases was affected after the onset of cold stress (Supplementary Table S3). Gene expression of a set of tRNAs for Ala, Ile, Leu, Ser, Glu and Met was also upregulated (Supplementary Table S3). In addition, the category 'ribosome' was upregulated as well (Supplementary Figure S1). Transcriptional level of genes required for the biosynthesis of many amino acids was previously found to be reduced during cold stress response in _B. subtilis_[95]. Induction of several genes encoding ribosomal proteins was also found in this bacterium [95]. However, in contrast to our results, genes encoding some of the tRNA synthetases, _aspS_, _hisS_, _metS_ and _thrS_, were repressed in _B. subtilis_[95]. Intriguingly, it was reported that psychrophilic strains of _Shewanella_ spp. and other cold-adapted \(\gamma\)-proteobacteria have a decreased alanine, proline, glycine and arginine content in their proteomes in comparison to the warm-adapted _Shewanella_, whereas the content of amino acids, such as lysine, isoleucine and asparagine was found to be increased in the cold-adapted proteins [96]. This characteristic amino acid profile was suggested to be beneficial for protein structural flexibility at low temperatures. In our study, we observed transcriptional decrease in expression of several amino acids metabolism-related genes, including those involved in the alanine, proline, glycine, and arginine metabolic pathways. Although lysine or isoleucine biosynthetic pathways in our study were found to be downregulated, we observed that the degradation pathway of these two amino acids was downregulated as well. It seems that in _S.