## 3 Results and Discussion
### Analysis of cotA and mhqO Gene Expression in B. safensis CN12 in the Presence of 4-n-NP  
Gene expression analysis is a widely known method to demonstrate the specific induction of genes; for this reason, many authors employ RT-qPCR to detect and quantify the expression of bacterial genes involved in the degradation pathways of organic pollutants in the environment [62; 63]. In this study, genomic, transcriptional, and quantitative approaches were employed to demonstrate the specific induction of two functional genes, _cotA_ and _mhqO_, which may play a key role in the 4-n-NP biodegradation pathway. The initial RT-qPCR amplification tests revealed unique fragments for both genes, confirming the primers' specificity. The tests also revealed that _cotA_ and _mhqO_ needed 10 ng of cDNA for effective amplification, while 16S rRNA could be amplified with as little as 0.003 ng of cDNA, resulting in CT values close to 20 for these genes. Regarding the qPCR amplifications conducted for the expression study, the slopes observed in the RT-PCR standard represented as regression line plots of the CT value vs. the log of the input cDNA were different for the studied genes (\(-4.36\), \(-3.62\), and \(-3.49\) for the _cotA_, _mhqO_, and _16S rRNA_ genes, respectively), which implies variable PCR efficiencies and excludes the applicability of the comparative CT method (\(\Delta\Delta\) CT method) [64]. This was confirmed by the validation test, which showed that both the target genes and the reference gene had different qPCR efficiencies, as the absolute value of the slope of the regression line between the log input RNA amount (ng) and \(\Delta\)CT was more than 0.1 in both cases. Therefore, both gene expressions were examined according to the Relative Quantitation of the Gene Expression approach by the standard curve method (Figure 3 and Table S3). The _cotA_ gene responds to 4-n-NP presence in _B. safensis_ CN12 and encodes a protein that shares similarities to the CotA protein of _B. pumilus_ A56. Gene induction corresponding to a transcription increase of \(\
eg 7.2\) times after 3 days of treatment was observed, coinciding with the point of maximum decrease in 4-n-NP concentration (Figure 3). From that moment on, higher expression continues to be observed at 5 and 7 days, but at lower values of \(\
eg 2.4\) and \(\
eg 1.8\) times, respectively, compared to the control (without 4-n-NP). In the case of the _mhqO_ gene, induction by 4-n-NP contact compared to samples in the absence of 4-n-NP was even more evident. Like the _cotA_ gene, the highest transcription rate of _mhqO_ was observed at 3 days, reaching values \(\
eg 13.9\) times higher compared to the control. Five days after inoculation, gene expression remained above the control (\(\
eg 1.8\) times). However, after 7 days, induction was no longer observed. (The level of transcription was \(\
eg 0.4\) lower than in the control.) Figure 3: Biodegradation of 4-n-NP in solution _by Bacillus safensis_ CN12 in the presence of glucose and the corresponding relative expression of _cotA_ and _mhqO_ (fold change compared to control). ANOVA: Comparison of _cotA_ and _MhqO_ gene expression between the control treatment (without 4-n-NP) and the treatment in the presence of 4-n-NP. Expression values are normalized to the 16S rRNA gene. Asterisks indicate statistically significant differences (\({}^{*}p<0.05\); \({}^{**}p<0.01\); \({}^{***}p<0.001\)). The _cotA_ gene was reported to code for a protein that belongs to the outer spore coat of _B. pumilus_ strain A56. This protein, known as CotA lactose, is a copper-dependent enzyme. It participates in the oxidation of a variety of substrates, including phenolic and non-phenolic compounds [59]. Based on the protein sequence and the transcription studies conducted, we can suggest that in the presence of 4-n-NP, this protein's expression in _B. safensis_ CN12 increases, enhancing the immobilization of 4-n-NP and subsequent oxidation on the bacterial cells. The MhqO enzyme is known to break the aromatic ring in organic compounds.