*3.3. Gene Expression Measurement by qRT-PCR*

The expression of *ompF*, *ompC*, *ompA*, *ompX*, *ompY*, *ompR*, *marA47*, and *marA48* genes in *Y. pseudotuberculosis* 488 was studied using qRT-PCR. Total RNA was isolated from the prepared bacterial pellets using the Aurum total RNA mini kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. The RNA concentration and purity were assessed by electrophoresis and a microvolume spectrophotometer (Implen GmbH, Munich, Germany). Of the purified total RNA, 2 μg was reverse-transcribed into cDNA with MMLV RT Kit (Evrogen, Moscow, Russia) and random hexamer primers (Evrogen, Moscow, Russia). The cDNAs were subsequently used to quantify the relative level of *ompF*, *ompC*, *ompA*, *ompX*, *ompY*, *ompR*, *marA47*, and *marA48* by qRT-PCR in Light Cycler 96 (Roche, Basel, Switzerland). The nucleotide sequences of the studied genes are listed in Table S2. The gene-specific primers used in this experiment are listed in Table S3.

RT-PCR was carried out with HS GoTaq Polymerase (Promega, Madison, WI, USA) and the dye Eva Green (Biotium, Fremont, CA, USA). The following thermal cycling parameters were used for the reaction: initial denaturation at 95 ◦C for 8 min; and 40 cycles of amplification: 95 ◦C for 15 s, 55 ◦C for 10 s, and 72 ◦C for 20 s followed by fluorescence reading. A melting curve was drawn at the end to evaluate the specificity of the PCR. Quantification for each target gene expression was determined by the 2−ΔΔCT method [60] using the 16S rDNA gene as a reference.

qRT-PCR was carried out on three independent biological replicates, each containing two technical replicates. The results are presented in Table S4 as mean ± SD. One-way ANOVA was performed to assess the differences between the means of the test and control groups, with a *p*-value of ≤ 0.05 considered significant.

#### *3.4. Porin Gene Sequencing*

The coding and regulatory parts of *ompF* and *ompC* genes were amplified using OmpF/C-Bam\_for and OmpF/C-Hind\_rev primers and sequenced with internal primers (Table S3) to cover all regions. DNA sequencing was done with an ABI 3130 xl automated sequencer (Applied Biosystems, Waltham, MA, USA) and the ABI Prism dye terminator sequencing kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer's directions.

#### *3.5. GFP Reporter System Construction*

The recombinant plasmids pACYC184-F-GFP and pACYC184-C-GFP containing the *Y. pseudotuberculosis ompF* and *ompC* regulatory regions fused to GFP were constructed as previously described [7]. To obtain the coding region of GFP, the plasmid DNA pTurboGFP (Evrogen, Moscow, Russia) was digested with *Bam*HI and *Hind*III (Fermentas, Waltham, MA, USA), and the resulting *gfp* fragment was dephosphorylated with CAP alkaline phosphatase (Fermentas, Waltham, MA, USA). The *ompF*/*ompC* promoter, and terminator regions were amplified using specific primers, which contained *Bam*HI and *Hind*III restriction sites, respectively (Table S3). The resulting PCR products were digested with the same endonucleases and ligated together (separately for *ompF* and *ompC*) with

*gfp* in a three-way ligation. Next, ligation mixtures were used as templates for amplifying *ompF*-*gfp* and *ompC*-*gfp* reporter constructions with OmpF-Bam\_for/OmpF-Hind\_rev or OmpC-Bam\_for/OmpC-Hind\_rev primers (Table S3). The PCRs were carried out using the following reagents: 2 U of GoTaq DNA polymerase (Promega, Madison, WI, USA), 1× buffer for GoTaq DNA polymerase, 0.2 mM dNTPs, 0.5 μM primers, and 25–50 ng of the template. The following reaction conditions were used: 1 cycle of 5 min at 95 ◦C for denaturation, 30 cycles of 20 s at 94 ◦C, 30 s at 55 ◦C, 1 min 30 s at 72 ◦C, and 1 final cycle of 5 min at 72 ◦C. The PCR fragments of expected lengths (1300–1400 bp) were cut from the agarose gel and phosphorylated with T4 polynucleotide kinase (Fermentas, Waltham, MA, USA). The resulting reporter cassettes were inserted in the dephosphorylated vector pACYC184 via *Eco*RV (Figure S1). Recombinant plasmids were transformed into *E. coli* DH5alpha strain by electroporation. The colonies were selected on LB agar plates supplemented with chloramphenicol. All constructs were verified by PCR and DNA sequencing with pACYCSal\_seq and pACYCEcV\_seq primers (Table S2). The final recombinant plasmids were introduced into competent cells of the *Y. pseudotuberculosis* 488 using electroporation.
