# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion
### Modified Expression of Genes Responsible for Membrane Integrity and Composition  
Properties of cellular membranes depend on the membrane structure and composition, influencing such important processes as transport of nutrients and ions, solute uptake, and osmotic pressure [81]. Proper membrane fluidity is required to maintain its structure and function. Thus, during cold stress, in addition to the response to the envelope stress by inducing sigma E-transcribed genes, bacteria have to deal with decreased membrane fluidity causing membrane stiffness [82]. Composition of the membrane fatty acids is one of most important features responsible for membrane fluidity. Bacteria respond to lowered temperature by increasing the proportion of unsaturated fatty acids (UFA) in the membrane, e.g., by desaturation of fatty acids in phospholipids or by incorporation of unsaturated instead of saturated fatty acids in the membrane structures [83; 84]. On the gene expression level, these adaptations can be achieved by various ways--in _E. coli_ and many other Gram-negative bacteria, the _lpxP_ gene is induced by stress and its product is responsible for attaching an unsaturated fatty acid (the palmitoleate) instead of a saturated laurate to lipid A [85]. In _Bacillus subtilis_, _Listeria monocytogenes_ and some other Gram-positive bacteria the proportion of branched fatty acids increases upon cold stress due to their elevated synthesis [86]; moreover, expression of the fatty acids desaturase gene, _des_, is induced [84; 87]. Synthesis of UFA can be achieved through two pathways--anaerobic, dependent on the FabA and FabB enzymes (as shown in _E. coli_), and aerobic--by the Des enzyme [84,88]. Expression of the _faba_ and _fabB_ genes was shown to be under control of an activator (FadR) and a repressor (FabR) [89]. Moreover, increased cold adaptation is correlated with the synthesis of polyunsaturated fatty acids (PUFA), with two main genes playing an important role in this process, _pks_ and _pfaB_[90]. Synthesis of UFA was described for _S. oneidensis_ and _S. putrefaciens_ (based on the presence of the FadR regulon and the Des enzyme), however, the PUFA synthesis was shown only for _S. oneidensis_ and _S. baltica_[91,92]. Our transcriptome analysis revealed that the genes involved in biosynthesis of UFA and PUFA are under cold stress mediated regulation. We found that expression of a _fabA_ ortholog (Shew185_2605) had increased after 180 min of the cold stress onset, which correlated with induced expression of _fadR_ (Shew185_1771) and decreased expression of _fabR_ (Shew185_2605) (Figure 6, Supplementary Figure S2 and Table S1). These observations indicate that the process of UFA synthesis is induced and upregulated as a response to cold stress. Analogous results were recently shown for _S. putrefaciens_[92]. Interestingly, expression of the _des_ gene (Shew185_2748) was decreased (Supplementary Figure S3 and Table S1) which again correlates with the _S. putrefaciens_ data, suggesting that the anaerobic pathway of UFA synthesis plays a major role in the cold-induced modification of membrane composition for the _Shewanella_ strains. PUFA production mediated by the _pks_ and _pfaB_ genes (Shew185_1419 and Shew185_1418, respectively) is also induced, as the expression of these genes was already elevated after 90 min of cold stress (Supplementary Table S1). Thus, our data present evidence that _S. baltica_ adjusts to the low temperature by maintaining its membrane fluidity by altered membrane composition.