# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR  
Total RNA was purified using the Total RNA Mini Plus kit (A&A Biotechnology, Poland) as per the protocol. The concentration of each sample was determined by analyzing 1.5 \(\upmu\)L of each sample on a NanoDrop Spectrophotometer. The TranScriba Kit (A&A Biotechnology, Poland) was used to perform the reverse transcription reaction following the manufacturer's protocols. RNA was purified from DNA using the Clean-Up RNA Concentrator Kit (A&A Biotechnology, Gdansk, Poland) and served as a template with a mixture of oligo (dT) 18 primers and random hexamers (in a 2:1 ratio). A negative reverse transcription control (no reverse transcriptase) was performed for each template. The prepared samples were incubated at 65 \({}^{\circ}\)C for 5 min and then cooled to 4 \({}^{\circ}\)C. Then, 4 \(\upmu\)L of reaction buffer, 2 \(\upmu\)L of dNTP's mix, and 4 \(\upmu\)L of TranScribaTM reverse transcriptase were added to each sample. After mixing the samples, they were incubated at 25 \({}^{\circ}\)C for 5 min and then another 30 min at 42 \({}^{\circ}\)C. To complete the reaction, the mixtures were heatedat 70 \({}^{\circ}\)C for 10 min. The cDNA prepared in this way was used as a template in the Real-Time PCR reaction. Real-Time PCR reactions were performed in a Stratagene Mx3000P thermal cycler (Agilent Technologies, Santa Clara, CA, USA), using the SYBR Green dye as a fluorochrome and the following program: 95 \({}^{\circ}\)C for 5 min, 40 cycles of 95 \({}^{\circ}\)C for 15 s, and 60 \({}^{\circ}\)C for 30 s. For each cDNA sample, a Real-Time PCR reaction was set up, amplifying fragments of 16S genes and the tested genes _catE_, _ctaD_, and _alupC_ for _P. putida_. Primer sequences used in qPCR are available in Table 2. PCR reactions, performed in triplicate, and the obtained Ct values were averaged. The specificity of each amplification was validated by examining its melting curve. Each run included a negative control. To create the comparative curve, cDNA solutions were prepared in a series of five-fold dilutions. The effects of pristine Al\({}_{2}\)O\({}_{3}\)NPs on gene expression are presented as relative changes in transcript levels of selected genes, normalized to the 16S rRNA reference gene and calculated using the 2\({}^{-\Delta\Delta\)Ct method. The \(\Delta\Delta\)Ct = \(\Delta\)Ct\({}^{\prime\prime}\)0\({}^{\prime\prime}\)\(-\)\(-\)\  
release potential in determining nanoparticle toxicity [38; 39]. Detailed toxicity data are provided in Supplementary Tables S1 and S3. Studies on other bacteria reveal varied responses to Al\({}_{2}\)O\({}_{3}\)NPs. For instance, _E. coli_ exhibited mild growth inhibition only at high concentrations (1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) [12]. Similarly, _P. stutzeri_ showed no significant toxicity but a dramatic 8.2-fold increase in _katB_ expression, indicating oxidative stress, while _Bacillus cereus_ was unaffected at the tested doses [13]. In contrast, Al\({}_{2}\)O\({}_{3}\)NPs were bactericidal against multidrug-resistant _S. aureus_ and coagulase-negative staphylococci [14]. Further supporting these observations, untreated wastewater samples stimulated bacterial growth in all tested variants, including control, nano-, and bulk-amended systems, likely due to the presence of readily bioavailable organic compounds. In the case of treated wastewater samples, no acute toxicity was observed according to ISO Guideline 10712-2 (1994) [32], as EC\({}_{50}\) values were equal to or greater than 100%. However, moderate growth inhibition was detected, ranging from 20% to 45% across tested samples, suggesting the presence of residual inhibitory substances despite the treatment process. These sublethal effects may result from residual organic and inorganic compounds that either interact with nanoparticles or act independently. Detailed toxicity data are provided in Supplementary Tables S2 and S4.