*2.3. Transcriptional Response of General Porin Genes to Prolonged Antibiotic Stress*

Several studies consider the modulation of porin gene expression as a rapid but temporary response of the cell to stress, which develops within the first 60 min [19,23]. However, in the case of antibiotic treatment, this mechanism can be replaced by more specific ones over time [1]. The data on the porin gene expression in time series are

important for a comprehensive understanding of *Y. pseudotuberculosis* defense against different antimicrobials.

To evaluate the effect of prolonged antibiotic exposure on the porin gene expression, *Y. pseudotuberculosis* 488 was cultivated in the presence of sub-MICs of these antibiotics for 16 h. In contrast to the early porin response, here we did not see significant changes in the expression of *ompF* and *ompC* genes in most samples treated with antibiotics (Figure 2). The data obtained indicate that transcription of general porins in *Y. pseudotuberculosis* cells transiently decreased within the first hour and subsequently returned to levels comparable to those of the untreated cells.

**Figure 2.** Relative expression levels of *Y. pseudotuberculosis ompF* and *ompC* after prolonged 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.

Previously, Viveiros et al. observed that *ompF* and *ompC* transcript levels in *E. coli* remained comparable to the untreated controls after prolonged exposure to tetracycline [24]. However, post-translational *marA*-mediated porin regulation to antibiotic stress has been shown to inhibit their synthesis.

Surprisingly, we found a 2.4- and 2.6-fold increase in the expression of *ompF* under prolonged tetracycline and chloramphenicol stresses at 27 ◦C, which no one had reported earlier. At the same time, incubating cells with these two antibiotics at 37 ◦C did not affect porin transcription. One of the possible reasons for the *ompF* upregulation in the presence of tetracycline and chloramphenicol could be the decreased expression of alternative porins.

We hypothesized that observed changes in the porin regulation under antibiotic stress over time could be caused by several factors. For example, stationary-phase bacteria must respond and adapt to a variety of environmental stresses (nutrient exhaustion, pH changes, oxygen limitation, and others). In such a case, the regulation of porins provides the balance of outer membrane permeability between self-defense and competitiveness [39,40]. The significantly reduced expression of general porins, protecting cells from antibiotics within the first hour, might entail high metabolic costs at the stationary phase, since vital nutrients are simultaneously excluded.

It should be noted that in addition to the transcriptional regulation of porin genes, post-transcriptional regulation might play a major role in the physiological adaptation to prolonged antibiotic exposure, as was mentioned previously [24].

Moreover, the acquisition of mutations in porin genes, leading to loss of proteins, size, and conductance modification of their channels or decreasing their expression level, is known as another common mechanism for reducing the sensitivity of bacteria to antibiotics. It was observed that porin-related mutations substantially influence resistance to β-lactams, fluoroquinolones, tetracycline, and chloramphenicol [1]. However, we performed *ompF* and *ompC* sequence analysis of *Y. pseudotuberculosis* samples after prolonged antibiotic treatment and did not reveal any changes within the porin-encoding parts and their regulatory regions.
