*2.4. ompR and marA Effect on Porin Response under Antibiotic Stress*

Next, we investigated the contribution of transcriptional factors in the regulation of porin genes.

The best-described input into controlling porin levels involves OmpR, which regulates *ompC* and *ompF* directly through OmpR–promoter DNA association [41]. The OmpR/EnvZ two-component system may play a major role in antibiotic resistance by modulating porin expression [42]. It was shown that OmpR deficiency in *Y. pseudotuberculosis* and *Y. enterocolitica* reduces bacterial sensitivity to the β-lactams, tetracycline, and polymyxin B, caused by the lack of OmpF and OmpC porins [43,44].

qRT-PCR analysis revealed differential expression of *ompR* in *Y. pseudotuberculosis* depending on the duration of antibiotic stress (Figure 3). Short exposure to antibiotics excluding carbenicillin and tetracycline at 27 ◦C slightly reduced (1.7- to 2.3-fold) its transcription associated with the downregulation of porin genes. On the contrary, prolonged antibiotic stress increased the level of *ompR* transcripts up to 5.3-fold; however, this did not affect the expression of *ompF* and *ompC*. A similar observation was described in the work of Viveiros et al., where the response of the *ompC* and *ompF* genes, despite the increased expression of *ompR*, remained comparable to their levels in *E. coli* cells not exposed to tetracycline [24]. Moreover, it was shown that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of the *Y. enterocolitica* strain to different chemical stressors [45]. It is tempting to speculate that a high level of *ompR* transcription in *Y. pseudotuberculosis* may be required to induce this efflux pump system as another general defense mechanism against antibiotics.

Multiple studies have shown that general porin expression is post-transcriptionally regulated by global regulator MarA, involved in multidrug resistance response during antibiotic stress [46,47]. *marA* locus can directly or indirectly increase the level of *micF*, a non-coding RNA stress response gene, causing a post-transcriptional downregulation of *ompF* mRNA and reducing the OmpF synthesis [47]. Two MarA-like proteins have been reported in *Yersinia pestis*: MarA47 and MarA48 [48,49].

To test their involvement in the regulation of *Y. pseudotuberculosis* porin expression under short- and long-term exposure to antibiotics, we measured the levels of *marA47* and *marA48* mRNA using qRT-PCR (Figure 3). We found different activities of *marA* transcripts depending on the time of antibiotic exposure. A short treatment of cells with the antibiotics caused upregulation of at least one *marA* gene, except for chloramphenicol, which did not affect the *marA* expression. In the case of long-term exposure to the tested antibiotics, the level of *marA* expression remained comparable to those of the control samples.

The obtained results led us to conclude that regulation of *Y. pseudotuberculosis* general porins mediated by MarA was observed only for early antibiotic stress caused by sublethal concentrations of tetracycline, kanamycin, and carbenicillin. During long-term antibiotic presence, *marA* transcription remained unaltered and therefore MarA did not play a significant role in porin regulation at the post-transcriptional level.

**Figure 3.** Relative expression levels of *Y. pseudotuberculosis ompR*, *marA47*, and *marA48* under antibiotic stress detected by qRT-PCR. (**A**) 1 h antibiotic exposure, (**B**) 16 h antibiotic exposure. 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.
