# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## 2 Results and Discussion  
### Global Changes in the Transcriptome of S. baltica in Response to Cold Stress  
To characterize the transcriptional response of _S. baltica_ to cold stress we exposed bacterial cells to 8 \({}^{\circ}\)C for 90 and 180 min, followed by RNA sequencing (RNA-seq). For selection of genes whose expression has significantly changed, we followed the rules of good practice in gene expression analysis [31]. The main role was to filter genes with distinct changes, therefore, our main data set was composed of genes which had at least a two-fold change in expression (\(\text{log}_{2}\text{FC}|>1\)) and adjusted \(p\) value < 0.01 (Supplementary Table S1). Due to the limited number of replicates, we decided to use these stringent filtering criteria as a minimum threshold for scoring differentially expressed genes in the first instance. The most prominent effect of cold stress was downregulation of gene expression (Figure 1A). With the above criteria, we identified 521 genes downregulated after the first 90 min of cold stress, out of which 491 genes were still downregulated after additional 90 min exposure to low temperature, indicating persistent downregulation (Figure 1B). In addition, 92 genes were downregulated exclusively after 180 min of cold stress. In contrast, only 216 genes were upregulated after 90 min, out of which 5 remained upregulated after subsequent 90 min of cold stress exposure (Figure 1C). Sixteen genes were upregulated exclusively only after 180 min of growth at 8 \({}^{\circ}\)C.  
In summary, the major effect of cold stress in _S. baltica_ is a massive decrease in gene expression, which was observed for approximately 70% of differentially expressed gene sets. The same direction in transcriptomic changes that was observed in our study, was reported for _S. oneidensis_[23] and _E. coli_[11] as well. Thus, downregulation of gene expression appears to be a general cold stress response in bacteria. Figure 1: Changes in the global gene expression upon cold stress. (**A**) Heatmap presenting changes in gene expression for significantly altered genes (\(\text{log}_{2}\text{FC}|>1\) and adjusted \(p\) value <0.01). Column 0 stands for the control sample; replicates for 90 and 180 min time points are labeled R\({}_{1}\) and R\({}_{2}\). Gene expression data are presented as Z-score transformed, scaled in rows values. (**B,C**) Venn diagrams representing overlap between differentially expressed genes at both time points. Since we performed the RNA sequencing experiment in duplicate, it raises a question about robustness and significance of our findings. As stated above, we used stringent criteria for finding genes with significant changes in expression. However, we are aware that despite high coverage (average 783\(\times\), Supplementary Table S2), the number of replicates allows for correct detection of differentially expressed genes with 40% probability (estimated with RNASeqPower package version 1.28.0 [32] for 783\(\times\) coverage, 2 replicates and \(\alpha=0.05\)). To verify our RNA-seq data, we tested the expression of 11 selected genes with quantitative reverse transcription PCR (RT-qPCR) assay (Figure 2). The comparison of gene expression patterns obtained from RNA sequencing and RT-qPCR assay revealed that all tested genes show the same direction of change (Figure 2C). This result is higher than the calculated 40% which is probably due to the stringent adjusted \(p\) value threshold used for filtering of our RNA-seq results. Nevertheless, our RT-qPCR analysis corroborates findings from the RNA-seq analysis. Figure 2: Validation of RNA sequencing results with RT-qPCR. (**A**) Log\({}_{2}\) fold change of relative gene expression measured by RT-qPCR. Data were normalized to three reference genes (for details see the Materials and methods, Section 3.4). Each point represents average of four technical repeats for a biological replicate. Red and blue colors mark 90 and 180 min time points, respectively.