## 2 Results
### TA System Dependent Stress-Response in a Bacterial Logarithmic Growth Phase  
To proceed with the study of stress factors in the activation of chromosomal TA systems, five type II TA systems that were detected in only one copy in BA1250 chromosome were selected: CcdA-CcdB, MazeF-MazF, PrIF-YhaV, YefM-YoeB, and PasI-PasT. For this reason, BA1250 bacterial samples were obtained from culture in the logarithmic phase under specified stress conditions, and the system's expression was quantified by qPCR. All qPCR reactions were performed to analyze the relative expression of these genes in comparison to LB cultures.  
In the bacterial logarithmic growth phase, there was a stress-response-dependent system, since the _yoeB-yefM_ genes were observed to be upregulated under conditions of nutritional starvation, whereas they were downregulated in the presence of osmotic conditions (Figure 2). The same osmotic condition led to an upregulation in the transcription of the _ccdB-ccdA_ genes. The _mazE-mazF_ genes were negatively regulated in response to acid stress, while oxidative conditions led to an exclusively downregulation of the antitoxins _mazE_ and _pasI_. The contrary was observed for the _yhaV-prIF_ genes since they were downregulated under conditions of oxidative stress, but only the antitoxin _prIF_ was downregulated in response to acid stress.  
Figure 2: Relative expression of toxin–antitoxins in a log growth phase in duplicates of three independent experiments. _E. coli_ BA1250 toxin–antitoxin gene pairs (_ccdB/ccdA, yhaV/prIF, mazE/mazF, yoeB/yefM,_ and _pasT/pasI_) were evaluated in the log growth phase under nutritional scarcity, oxidative stress, acid shock, osmotic stress, and LB medium condition. Genes were considered up/downregulated when relative average expression was \(-1>\text{Log2 FC}>1\) in comparison to the LB group. Statistical significance was considered when the \(p\) value < 0.05 in 2-way ANOVA test comparing toxin and antitoxin at the same condition. (*) Represent statistical significance with \(p<0.05\): (**) represent statistical significance with \(p<0.01\).  
Nutritional Starvation and Acid Environment Activate TA Systems in the Bacterial Stationary Growth Phase  
The assessment of toxin-antitoxin system expression was also performed in the stationary growth phase where in all qPCR reactions the relative expression of the genes was compared to LB cultures. The expression levels of the _ccdB-ccdA_ and _mazE-mazF_ genes did not exhibit any statistically significant variation under the tested stress conditions during the stationary phase (Figure 3). The same occurred under osmotic and oxidative stresses since it did not activate any of the selected TA systems. In contrast, it was observed that the acid environment had a massive influence on the TA system, since _yhaV-prIF_, _yoeB-yefM_, and _pasT-pasl_ genes were activated in this condition. Besides the acid environment, the _past-pasl_ genes and the toxin _yhaV_ were also active during nutritional starvation.  
In specific stress conditions, differences were observed in the expression of only one component of the TA system, either toxin or antitoxin genes. It was evident that most genes in the TA systems were expressed concomitantly. However, notable differences were observed during the log phase for the _yhaV-prIF_ genes (in an acid environment) and the _past-pasl/mazE-mazF_ genes (under oxidative stress). In the stationary phase, a scenario emerged where all systems were expressed similarly, except for the YhaV-PrIF system.