# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion
### Downregulation of a Type II Secretion System in Response to Cold Stress  
All bacterial species transport a great number of proteins across the cytoplasmic membrane, from the cytosol to specific outer locations. Lipoproteins, periplasmic proteins, cell wall-associated proteins, outer membrane or extracellular proteins are all subjected to secretion in growing cells [45]. Processing and translocation of preproteins are pointed out to be reduced in the early phase in bacteria exposed to cold stress conditions, as well as in the cold adapted cells [45]. Secretion system in the Gram-negative bacteria consists of several distinct systems mediating secretion of proteins for example involved in biogenesis of pili and flagella, nutrient acquisition, virulence, adherence and biofilm formation, or efflux of drugs and other toxins [64; 65]. One of these systems, the type II secretion system (T2 SS), is responsible for translocation of exoproteins through the outer membrane into the extracellular _milieu_. In _S. oneidensis_ and _Shewanella putefaciens_, this system is directly involved in extracellular translocation of outer membrane cytochromes, as well as exoproteins engaged in dissimilatory reduction of insoluble metal oxides, electron transport and anaerobic respiration [66; 67]. The multi-component T2S system consists of a cytoplasmic ATPase, several inner membrane proteins, a periplasmic pseudopilus and a secretin pore embedded in the outer membrane [68] (Figure 4). Components of this system belong to a family of general secretory pathway (Gsp) proteins found among all bacterial species. Secretion of exoproteins with T2 SS is a two-step process [69]. First, the substrate exoprotein must be translocated across the inner membrane by either SecA and signal recognition particle (Sec-SRP) translocon, or the twin-arginine targeting (Tat) translocon. Second, after reaching the periplasm, the exoprotein is exported by the T2S system across the outer membrane. Figure 3: Expression changes in _S. baltica_ flagellar assembly pathway upon cold stress. Scheme of flagellar assembly pathway (source: Kyoto Encyclopedia of Genes and Genomes (KEGG) database). Tripartite heatmap boxes show row Z-score scaled normalized expression values for corresponding genes upon cold stress at 0, 90 and 180 min time points, respectively. Our transcriptomic study revealed that in _S. baltica_, the T2S and Sec-SRP system components are regulated in response to cold stress exposure (Figure 4). We found that 11 genes of the large Gsp operon encoding T2 SS components were significantly downregulated after 90 and 180 min of temperature downshift. The observed downregulation affected genes coding for all components of the T2S system, including the GspD secretin (Shew185_0149), all inner membrane proteins (IMP)--GspC, GspF, GspG, GspH, GspI, GspJ, GspK, GspL and GspM (Shew185_0148, Shew185_0151, Shew185_0152, Shew185_0153, Shew185_0154, Shew185_0155, Shew185_0156, Shew185_0157 and Shew185_0158, respectively) and the GspE ATPase (Shew185_0150), and the extent of downregulation was greater over time (Figure 4). Our results indicated that the entire T2 SS is affected by low temperature and this may probably lead to a serious reduction in protein secretion mediated by this system. To the best of our knowledge, downregulation of type II secretion system in response to cold stress was not previously reported for the _Shewanella_ genus. The translocation of unfolded proteins from the cytoplasm to periplasm, where the folding occurs before the protein reaches the T2S system, is mediated by the Sec-SRP translocase system located in the inner cell membrane. One key element of this process is the SecB chaperone responsible for post-transcriptional delivery of preproteins to the Sec translocon [69]; the other one is SRP, composed  
Figure 4: Expression changes affecting the _S. baltica_ type II secretion system upon cold stress.