*3.6. Flow Cytometry*

To obtain comparable measurements with flow cytometry, the *Y. pseudotuberculosis* 488 containing recombinant plasmid pACYC184-F-GFP or pACYC184-C-GFP were grown in the same conditions as for qRT-PCR analysis. All cultures were incubated in 96-well plates without/with a sublethal concentration of antibiotics (Tet, Km, and Cb) and the addition of chloramphenicol (10 μg/mL) at 37 ◦C and 27 ◦C with shaking. After 16 h of cultivation, the cells were washed with PBS, pH 7, and diluted to an OD600 of 0.01. The flow cytometry measurements were made with a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). The excitation beam for the GFP was set at 488 nm, and the emission signal was captured with a 530/30 nm bandpass filter. The signals were amplified with the logarithmic mode for SSC, FSC, and fluorescence. For each sample, 100,000 events were recorded. The data were captured with the BD FACSdiva software (version 6.1.2) (BD Biosciences, San Jose, CA, USA), and for data analysis FCS Express 6 (De Novo Software Inc., Los Angeles, CA, USA) was used. The mean fluorescence value of the whole population was chosen as a global indicator of porin expression level in each bacterial culture. *Y. pseudotuberculosis* 488 without plasmids was used as a negative control to exclude background fluorescence.

#### **4. Conclusions**

In summary, we revealed temporal regulation of *Y. pseudotuberculosis* generalporin genes *ompF* and *ompC* caused by sublethal concentrations of different antibiotics. The porin transcription initially decreased, providing early defensive response of the bacterium, while it returned to that of the untreated cells on prolonged antibiotic exposure. Unlike the major porin genes, the transcription of the alternativeporin genes *ompX* and *lamB* was increased. Such modulation of porin transcription reflects the model of *Y. pseudotuberculosis* physiological adaptation to antibiotic exposure.

The main finding was a phenotypic heterogeneity of *Y. pseudotuberculosis* population manifested in variable porin gene expression under carbenicillin exposure. To verify whether such phenotypic heterogeneity offers a competitive advantage and contributes to low carbenicillin susceptibility for a particular *Y. pseudotuberculosis* subpopulation, future research is warranted.

**Supplementary Materials:** The following are available online, Figure S1: GFP reporter constructions; Figure S2: GFP fluorescence intensity in *Y. pseudotuberculosis* 488 transformed with *ompC/ompF* promoter-fused GFP reporter constructs under increasing Cm exposure; Figure S3: GFP fluorescence intensity in *Y. pseudotuberculosis* 488 transformed with *ompF/ompC* promoter-fused GFP reporter constructs at 27 ◦C and 37 ◦C incubation temperature; Table S1: MIC of antibiotics against *Y. pseudotuberculosis* 488; Table S2: Nucleotide sequences of *Y. pseudotuberculosis* 488 genes used in the study; Table S3: Oligonucleotide primers used in the study; Table S4: Average Ct values for *ompF*, *ompC*, and 16S rRNA of *Y. pseudotuberculosis* 488.

**Author Contributions:** Conceptualization, M.I. and A.R.; methodology, M.I., A.S., N.C., and E.B.; software, E.B.; investigation, E.B., N.C., K.G., and A.S.; resources, M.I. and A.R.; data curation, M.I.; writing—original draft preparation, E.B. and M.I.; writing—review and editing, M.I. and A.R.; supervision, M.I.; funding acquisition, M.I. and E.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Unpublished data are available from the authors.

**Acknowledgments:** We are grateful to the anonymous reviewers and editors that have improved our manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

