# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
To better understand the potential mechanisms underlying the observed toxic effects, changes in the expression of oxidative stress-related genes were also analyzed. Alterations in gene expression were observed as early as 1 and 16 h after _P. putida_ exposure to Al\({}_{2}\)O\({}_{3}\) nanoparticles and differed from the responses under control conditions. A significant, time-dependent increase in the expression of all three analyzed genes was observed. After 1 h, _katE_ and _alhpC_ expression increased approximately 2.5-fold compared to the control, while _ctaD_ showed only a slight elevation (fold change \(\sim\)1.2) (Figure 1A). After 16 h, _katE_ expression reached nearly 4.5-fold and _alhpC_ approximately 3.9-fold, indicating a sustained and intensified oxidative stress response. The expression of _ctaD_ also increased to around 3.0-fold, suggesting that prolonged nanoparticle exposure may trigger broader cellular responses, including those related to energy metabolism and electron transport (Figure 1B). In the presence of the bulk form of the material, changes in gene expression were less pronounced, although still detectable. After 1 h of exposure, moderate upregulation of _katE_ and _alhpC_ was observed (\(\sim\)1.6 and \(\sim\)1.5, respectively), while _ctaD_ expression was slightly decreased compared to the control (Figure 1A). After 16 h, _katE_ and _alhpC_ expression levels increased to approximately 2.0 and 1.8, respectively, reflecting a moderate activation of the stress response. The expression of _ctaD_ also increased (\(\sim\)1.5), although it remained significantly lower than in the nanoparticle treatment (Figure 1B). The expression of the stress-related genes _katE_, _ctaD_, and _alhpC_ was also evaluated in bacterial cells exposed to untreated (I) and biologically treated (E) wastewater samples supplemented with either Al\({}_{2}\)O\({}_{3}\)NPs, bulk material, or no additives (control) (Figures 2-4). In untreated wastewater containing Al\({}_{2}\)O\({}_{3}\) nanoparticles (WW-NPs-I), all three genes showed consistently elevated expression throughout the 63-day experiment. For _katE_, expression levels remained above 4.0 on day 1 and gradually declined to approximately 3.3 by day 63 (Figure 2). _AhpC_ followed a similar pattern, starting at \(\sim\)3.6 and decreasing to \(\sim\)3.0 (Figure 3), while _ctaD_ expression ranged from \(\sim\)2.2 on day 1 to \(\sim\)1.7 by the final time point (Figure 4). These results indicate a sustained oxidative stress response triggered by the presence of nanoparticles in the raw wastewater matrix. In the untreated wastewater control samples (WW-Control-I), an initial moderate increase in gene expression was observed (e.g., _katE_\(\sim\)3.5, _ctaD_\(\sim\)2.0, _alhpC_-3.2), likely due to the presence of bioavailable organic matter and associated metabolic activity. However, gene expression gradually decreased over time, reaching baseline or near-baseline levels by day 63 (_katE_\(\sim\)1.4, _ctaD_\(\sim\)1.0, _alhpC_-1.3),indicating the transient nature of the initial response in the absence of particulate additives (Figures 2-4). In biologically treated wastewater samples containing nanoparticles (WW-NPs-E), elevated gene expression was still observed, although at consistently lower levels than in the untreated equivalents. Expression of _katE_ decreased from \(\sim\)4.1 to \(\sim\)3.2 over the course of the experiment, _alpC_ from \(\sim\)3.4 to \(\sim\)2.8, and _ctaD_ from \(\sim\)2.0 to \(\sim\)1.6. These data suggest that biological treatment reduced but did not eliminate the biological activity or bioavailability of nanoparticles (Figures 2-4). Gene expression analysis in _P. putida_ revealed the molecular response to stress induced by exposure to aluminum oxide nanoparticles, showing time-dependent expression patterns across all analyzed genes. Following exposure to pristine Al2O3 NPs, significant upregulation of _katE_ and _alpC_--encoding catalase and alkyl hydroperoxide reductase, respectively--was observed.