*2.2. An Early Transcriptional Response of General Porin Genes to Antibiotic Stress*

Rapid modulation of porin gene expression is the first line of bacterial defense against harmful compounds, including antibiotics, which increases the chances of survival and adaptation of microorganisms to stressful conditions [19].

To study the early porin response in *Y. pseudotuberculosis* 488, we treated bacterial cells by sub-MICs of antibiotics for an hour, followed by measuring the level of *ompF* and *ompC* transcripts using qRT-PCR. The results obtained (Figure 1) indicate that kanamycin and tetracycline cause a transcription decrease in both general porins, regardless of temperature. *ompF* and *ompC* expression were downregulated 2.3- to 4-fold and 2.2- to 4.4-fold, respectively, in the presence of these antibiotics. It is not surprising because the reduced porin expression is considered to be a common strategy for protection against antibiotics. However, tetracycline and kanamycin downregulated only OmpF synthesis and upregulated OmpC production in *Escherichia coli* [30–33]. Nevertheless, Agafitei et al. have

shown that reduced expression of the OmpC porin contributes to kanamycin resistance [34]. Viveiros et al. also found a coupled downregulation of the *E. coli* OmpF with the OmpC during exposure to increasing concentrations of tetracycline [24].

**Figure 1.** Relative expression levels of *Y. pseudotuberculosis ompF* and *ompC* after short-term antibiotic exposure detected by qRT-PCR. (**A**) 27 ◦C incubation temperature; (**B**) 37 ◦C incubation temperature. All results are expressed as mean ± standard deviation from three independent experiments. An asterisk indicates *p*-value < 0.05 vs. respective untreated control. Significance was calculated using one-way ANOVA. Km—kanamycin, Tet—tetracycline, Cb—carbenicillin, and Cm—chloramphenicol.

Interestingly, two other antibiotics caused different transcriptional responses of the porins in *Y. pseudotuberculosis* 488. After one-hour exposure of cells to carbenicillin, the expression level of *ompF* showed a 2.3- to 3-fold decrease, while the expression of *ompC* was not changed significantly (Figure 1). This result supports the findings that OmpF is the preferred route of entry for β-lactams [35]. We suppose that a decrease of *ompF* mRNA level observed at both temperatures could be a protective response of *Y. pseudotuberculosis* cells to the carbenicillin stress. This opinion agrees with the previous finding that lack or inhibition of OmpF contributes to increased resistance to β-lactams [36,37]. It was also unexpected that under short-term chloramphenicol stress, the *ompF* expression was not significantly altered, whereas the *ompC* expression was downregulated 3.9 to 4.6 times (Figure 1). Chloramphenicol has been reported, unlike β-lactams, to utilize both porins equally well [16]. However, there is no observation to indicate any effect of a reduced level of OmpC alone on the penetration of this antibiotic [38].

Considering the qRT-PCR results, we assumed that the general porins OmpF and OmpC of *Y. pseudotuberculosis* are involved in the early response to various antibiotic stresses caused by sublethal concentrations and provide the cells with the first defense mechanism by reducing the transcription level of one or both of their genes depending on the antibiotic type.
