# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion
### Upregulation of the Sulfur Assimilatory Pathway upon Cold Stress  
Sulfur is an element essential not only for bacteria, but for life in general. Various oxidation states of sulfur--ranging from (+6) in sulfate SO42-, through (+4) in sulfite SO32-, to (-2) in sulfide H2S, implicate the vast reduction/oxidation potential of this element. In general, sulfur metabolism comprises of sulfur oxidation and sulfur reduction pathways. The sulfur oxidation pathway, also known as the sulfur oxidation pathway (SOX) system, is found in photosynthetic and non-photosynthetic sulfur-oxidizing bacteria [71]. The sulfur reduction pathways comprise of two contradictory metabolic routes leading to either assimilation or dissimilation of sulfur-rich compounds. This attribute accounts for the great potential of bacteria to adapt their metabolism to distinct or changing environmental conditions [72; 73; 74]. In our study, we found that the transcriptional response of _S. baltica_ to cold stress includes significant upregulation of expression of genes assigned to the assimilatory branch of the sulfate reduction pathway (Figure 5). In general, this process is energy consuming and produces reduced sulfur compounds which are further utilized for biosynthesis of sulfur-containing amino acids. After 90 min of cold stress, we observed upregulation of three genes involved in this pathway encoding three subunits of the ABC sulfate transporter--the ATPase (Shew185_1063) and two inner membrane subunits (Shew185_1064 and Shew185_1065). Upregulation of these genes was still present after 180 min of the onset of cold stress. The sulfate ABC transporter is involved in import of sulfates from the environment. In the assimilatory pathway, sulfate is activated with ATP by the CysND sulfate adenylyltransferase (Shew185_0928, Shew185_0929,) and forms adenylyl sulfate (APS), which is next converted into 3'-phosphoadenylyl sulfate (PAPS) by the CysC adenylyl-sulfate kinase (Shew185_0931). PAPS is then reduced to sulfiteby the CysH phosphoadenosine phosphosulfate reductase (Shew185_0921). Finally, sulfite (SO3\({}^{2-}\)) is reduced to sulfide (H2S) by the CysI) assimilatory sulfite reductase (Shew185_0919) [72; 73; 74; 75; 76]. All of the genes encoding these enzymes were significantly upregulated in _S. baltica_ after 90 and 180 min of cold stress (Figure 5). Hydrogen sulfide (H2S) is not directly excreted, but is used to synthesize L-cystine (with accompanying acetate release) and/or L-homocysteine (with accompanying succinate release). Figure 5: Expression changes in _S. baltica_ sulfur metabolism processes upon cold stress. (**A**) Simplified scheme of sulfur assimilatory pathway and a fragment of serine, homoserine and cysteine metabolism pathways are presented (based on: KEGG database). Tripartite heatmap boxes, fixed on affected pathways (full black circles), show row Z-score scaled normalized expression values for corresponding genes upon cold stress at 0, 90 and 180 min time points, respectively. Names of specific enzymes or subunits are shown next to the relevant gene names (black boxes). Yellow circles represent sulfur-containing compounds. Oxidation states and formulas (in yellow) are shown for key inorganic sulfur compounds—sulfate, sulfite and sulfide. (**B**) 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 R1 and R2. Genes encoding enzymes catalyzing these reactions, CysK (Shew185_2117) and MetB (Shew185_0536) were also upregulated in cold stressed _S. baltica_. It is interesting that the only downregulated genes in the sulfur metabolic pathway were those encoding the tetrathionate reductase--_trB_ (Shew185_3734), and thiosulfate/3-mercaptoethanolpyruvate sulfurtransferase (Shew185_1160), i.e., enzymes playing a key role in tetrathionate and thiosulfate metabolism.