# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
These enzymes are key components of the oxidative stress response, responsible for the detoxification of reactive oxygen species (ROS). The most pronounced changes occurred after 16 h, indicating sustained oxidative pressure in bacterial cells. Similar induction of these genes has been reported in other Gram-negative bacteria exposed to TiO2 NPs and ZnONPs [40; 41]. Figure 1: Relative expression levels of _katE_, _ctaD_, and _alpC_ genes in _Pseudomonas putida_ exposed to aluminum oxide nanoparticles (Al2O3 NPs), bulk Al2O3, and control conditions. (**A**) Gene expression after 1 h of exposure. (**B**) Gene expression after 16 h of exposure. The results indicate time-dependent transcriptional responses associated with oxidative stress and energy metabolism pathways, with distinct differences between nanoparticulate and bulk forms of Al2O3. Figure 3: Effects of influents and effluents on the expression of _ahpC_ in _P. putida_ following 16 h exposure. Data are expressed as fold induction relative to the untreated control (artificial water) and were normalized to the 16 rRNA gene. Dotted lines indicate expression differences between treatments. Figure 2: Effects of influents and effluents on the expression of _katE_ in _P. putida_ following 16 h exposure. Data are expressed as fold induction relative to the untreated control (artificial water) and were normalized to the 16 rRNA gene. Dotted lines indicate expression differences between treatments. The expression of _ctaD_, encoding a subunit of cytochrome oxidase--a central element of the respiratory chain--was also elevated. This may reflect disruptions in membrane-associated energy metabolism or increased cellular energy demand under stress conditions. However, the correlation between _ctaD_ expression and ATP synthesis is complex, as nanoparticle-induced membrane damage or the presence of uncoupling agents in wastewater could lead to proton leakage across the bacterial membrane, dissipating the proton motive force (PMF) without driving ATP synthase activity [42]. In such scenarios, elevated _ctaD_ expression may serve to maintain electron flow and oxygen consumption to counteract stress, rather than directly contributing to ATP production. Comparable changes were previously observed in _P. putida_ exposed to hematite nanoparticles (Fe\({}_{2}\)O\({}_{3}\) NPs), which reduced the expression of _fumC_ and _sodA_, pointing to metabolic imbalance and oxidative stress. Downregulation of _fumC_, coding for fumarase C in the TCA cycle, suggests reduced NADH production and impaired electron transport, while decreased _sodA_ expression may lead to ROS accumulation and respiratory chain dysfunction [43]. Additionally, [PERSON] et al. (2006) [44] demonstrated that inactivation of the cyo-type ubiquinol oxidase in _P. putida_ affects the global regulation of genes involved in electron transport and energy metabolism. While not directly related to nanoparticle exposure, this finding supports the notion that alterations in electron transport components can modulate bacterial adaptation to environmental stressors [44]. Our results show that both raw and treated wastewater containing Al\({}_{2}\)O\({}_{3}\)NPs induced changes in _katE_, _alpC_, and _ctaD_ expression in _P. putida_, with the strongest response observed in raw wastewater. The expression profiles suggest that nanoparticle exposure in a complex wastewater matrix may trigger both acute oxidative stress responses and longer-term metabolic adaptations involving electron transport. In raw wastewater samples, the marked upregulation of _katE_ and _alpC_ points to strong activation of cellular defence systems for detoxifying hydrogen peroxide and organic hydroperoxides. Al\({}_{2}\)O\({}_{3}\)NPs that are present in raw wastewater in a more reactive, unmodified form may have induced excessive ROS  
Figure 4: Effects of influents and effluents on the expression of _ctaD_ in _P. putida_ following 16 h exposure.