# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion
### Transcriptional Reprogramming to Adjust to Cold Stress  
Transcriptional regulation in bacteria is typically controlled at the transcription level, which would allow the quickest response with a minimum energy loss. Thus, bacteria have developed numerous strategies to regulate transcription initiation in response to various challenges. One of the most efficient ones is using various sigma subunits of the RNA polymerase to alter the transcription machinery's capacity and redirect its functioning towards specific promoters, operons and regulons. Some sigma factors are directed to counteract and protect bacteria from the environmental challenges, such as heat stress, nutrient limitations and others, and their number usually correlates with the variability of habitats and metabolic flexibility [34]. Transcriptional reprogramming through various sigma usage would be a sign of bacteria dealing with stress conditions. In fact, we observed that the gene encoding the housekeeping sigma 70, _rpoD_ (Shew185_3128), was downregulated upon cold stress, as was the case for genes coding for sigma 32 (Shew185_4161), sigma 54 (Shew185_0686) and sigma E (Shew185_1239--_rpoE_). A significant upregulation was observed for the Shew185_2636 gene coding for the extracytoplasmic function (ECF) subfamily RNA polymerase sigma 24 factor (_rpoE2_) and for Shew185_2919--the flagellar biosynthesis sigma factor (Supplementary Table S1). Changes in gene expression revealed by the transcriptome analysis were confirmed for genes chosen for further qPCR verification: _rpoE_, _rpoE2_ and _rpoD_ (Figure 2). Notably, for the general transcription response, expression of the gene responsible for synthesis of the stringent response alarmones, ppGpp and pppGpp, had increased (Supplementary Table S1). The possible accumulation of (p)ppGpp would result in favoring of alternative sigma factors' association with the RNA polymerase core for upregulation of stress-induced gene expression; in case of the studied cold stress--this would mainly concern the ECF factors and flagellar sigma factors. ECF sigma factors are responsible for integrating signals from the outer membrane and its components, such as proteins (transporters, porins) and lipopolysaccharidesaccharides, as well as changes in the concentrations of compounds outside the cells [35; 36; 37]. Their response to the envelope stress is often regulated by the numerous anti-sigma factors which, being anchored as transmembrane proteins, serve as signal transduction systems, releasing sequestered ECF sigma factors to activate relevant regulons [38; 39]. Interestingly, expression of one of the annotated _S. bultica_ anti-sigma E factors, RseA (Shew185_1240), was downregulated (Supplementary Table S1). Many bacteria with more complex life styles have multiple ECF sigma factors, which together with their regulators constitute a complicated control system [38]. In the _Shewanella_ genus, the ECF factors were described so far only for _S. oneidensis_, where sigmas from the ECF-like family were identified [40]. The two _S. bultica_ ECF sigma factors that were observed in our analysis to have altered expression upon cold stress are S. _oneidensis rpoE_ and _rpoE2_ orthologs (Shew185_1239 and Shew185_2636, respectively). The _S. oneidensis rpoE_ was described as a sigma factor responsive to high temperatures and osmotic stress, while _rpoE2_ was not reported to be required for a temperature stress but was involved in the oxidative stress response [40]. One of the genes transcribed by the RpoE2-RNA polymerase, the periplasmic glutathione peroxidase, is important for resistance to the oxidative stress. In our studies, _S. bultica_ Shew185_3037 gene coding for the periplasmic glutathione peroxidase and Shew185_3036 encoding a ferrochelatase, present in the same operon, displayed an increased transcription, corresponding with _rpoE2_ upregulation. These data indicate that upon cold stress _S. bultica_ responds by inducing mechanisms for protection against oxidative stress.