# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion
### Expression of Flagellar Assembly-Related Genes in Cold Stress Exposed S. baltica  
Bacterial motility is one of the key features to ensure a proper response to environmental stressors. The most common organelles in motile bacteria are flagella, necessary not only for chemotaxis but also for adherence, biofilm formation, virulence or such processes as reduction of insoluble minerals [48; 49; 50; 51]. A typical flagellum structure is well described and conserved among bacteria, and consists of over 20 proteins forming specific structures: the hook and basal body that anchor the flagellum to the membrane, and the external filament [52; 53; 54]. The energy that bacteria use to produce flagella is substantial, as it requires, among others, synthesis of thousands of the flagellin molecules to build a filament; moreover, action of the flagellum is based on the proton pumps serving as motor force generators. Such a high energy usage comprises a large burden on bacterial metabolism, and thus the process of flagellar assembly, as well as its rotation, requires a precise regulation [55]. This is achieved by a sequential cascade of gene transcription. In bacteria with a polar flagellum, the first level of transcription control is usually mediated by promoters dependent on sigma 54 and transcription regulators (among others, sigma subunit specific for flagella). Subsequent expression tiers are under control of these factors and encode flagellar structural genes [56]. Bacteria of the _Shewanella_ genus, although highly diverse in terms of habitats, are usually motile with one polar flagellum. As described for the group representative, _S. oneidensis_, most components of the flagellum (except for the motor protein genes) are encoded by genes located in one chromosome segment [57]. A similar genomic set-up is present in _S. baltica_ (based on the sequenced genomes). We observed that the flagellar structural genes (encoding the basal body, rod, filament, as well as the anchoring complex) were upregulated in _S. baltica_ after the onset of cold stress, with higher expression usually at the 90 min time point (Figure 3). Such an increased expression was also noted for the regulatory genes, e.g., the flagellar biosynthesis sigma factor, _fliA_. The temperature dependent induction of flagellar synthesis has been reported for various bacteria, including pathogenic _Listeria monocytogenes_[58], where the Csp proteins regulate the flagellum gene expression [59], and also in _Yersinia enterocolitica_[60]. Flagellar assembly was induced in the extremophile bacterium, _Thermoaneraobacter tengcongensis_[55] as part of a cold stress response; also, in an Antarctic bacterium, _S. livingstonensis_, cold adaptation was accompanied by flagellar genes' overexpression [24]. Notably, the increased expression of genes encoding flagellar proteins was also observed in _E. coli_ after a temperature downshift [61]. These data indicate that the motility of cold exposed flagellated bacteria intensifies, based on the upregulation of transcription of flagellar genes and those involved in the flagellar assembly. Interestingly, this adaptation does not seem to correlate with the bacterial life style or habitat. As observed by our transcriptomics data, _S. baltica_ follows this pattern; the RT-qPCR analysis confirmed this observation for the flagellar basal body rod protein-encoding gene (_flgF_) whose expression level was increased at 180 min time point, while the _fliA_ transcript level was elevated mostly at 90 min, which corresponds with the role of _fliA_-encoded sigma factor to promote the flagellar gene expression (Figure 2A). A biological sense of the induced flagella production and bacterial motility during temperature decrease has been proposed to be related to the need for an increased chemotaxis towards energy sources, the usage of unusual electron acceptors, and also as a response to higher viscosity of liquids in the cold [58; 62; 63]. Besides that, flagella promote biofilm formation, which can be also important for survival of _S.