*2.5. Transcriptional Response of Y. pseudotuberculosis Alternative Porins to Prolonged Antibiotic Stress*

In addition to major porins, *Enterobacteriaceae* can express alternative outer membrane proteins that may contribute to the adaptation of bacteria to antibiotic stress by modulating OmpF and OmpC levels or directly changing the cell permeability [35,50].

Therefore, to better understand the mechanism of *Y. pseudotuberculosis* adaptation, we investigated the transcriptional response of the genes coding for OmpX, OmpA, LamB, and OmpY porins to prolonged antibiotic stress.

Using qRT-PCR, we observed the increased activity of *ompX* transcripts (1.7–3.2-fold) in the presence of most antibiotics (Figure 4), which is consistent with previously reported results [50]. It is believed that overexpression of OmpX may impair the normal assembly of major porins, which leads to their subsequent degradation by Deg proteases [51]. From this, we hypothesized that OmpF and OmpC expression of *Y. pseudotuberculosis* could be regulated by this mechanism at the post-translational level.

We also observed the induction of *lamB* transcription in the presence of kanamycin, carbenicillin, and chloramphenicol at 37 ◦C (Figure 4), when its initial mRNA level was significantly lower compared to 27 ◦C (data not shown). It is interesting to note that overexpression of LamB previously found for *Enterobacter aerogenes*-resistant isolates was associated with a low amount of its major porins [52].

The transcription of *ompA*, playing a more important role in the maintenance of membrane integrity, rather than antibiotic transport [35], remained unaltered in *Y. pseudotuberculosis*, except for chloramphenicol exposure, where the *ompA* expression increased up to 2.5-fold (Figure 4).

**Figure 4.** Relative expression levels of *Y. pseudotuberculosis ompX*, *lamB*, *ompA*, and *ompY* under prolonged antibiotic stress detected by qRT-PCR. 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, we described a new quiescent porin OmpY in *Y. pseudotuberculosis* initially predicted from the genomic data [53]. To date, our knowledge of such porins has been very limited. Both the functional role of OmpY and the conditions significantly affecting its expression in *Yersinia* are still unknown. qRT-PCR analysis revealed *ompY* upregulation under chloramphenicol and tetracycline exposure at 27 ◦C, while at 37 ◦C its level did not change (Figure 4). Several studies have shown that the expression of quiescent porins was induced by antibiotics; however, the impact of such regulation on antibiotic susceptibility remains questionable [20,54].
