Differential Expression of Yersinia pseudotuberculosis General Porin Genes during Short- and Long-Term Antibiotic Stresses.

Here, we investigated general porin regulation in Yersinia pseudotuberculosis 488, the causative agent of Far Eastern scarlet-like fever, in response to sublethal concentrations of antibiotics. We chose four antibiotics of different classes and measured gene expression using qRT-PCR and GFP reporter systems. Our data showed temporal regulation of the general porin genes ompF and ompC caused by antibiotic stress. 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 alternative porin genes ompX and lamB was increased. Moreover, a short-term ompR- and marA-mediated porin regulation was observed. The main finding was a phenotypic heterogeneity of Y. pseudotuberculosis population manifested in variable porin gene expression under carbenicillin exposure. This may offer adaptive fitness advantages for a particular bacterial subpopulation.

Genome-Wide Analysis of PL7 Alginate Lyases in the Genus Zobellia.

We carried out a detailed investigation of PL7 alginate lyases across the Zobellia genus. The main findings were obtained using the methods of comparative genomics and spatial structure modeling, as well as a phylogenomic approach. Initially, in order to elucidate the alginolytic potential of Zobellia, we calculated the content of polysaccharide lyase (PL) genes in each genome. The genus-specific PLs were PL1, PL6, PL7 (the most abundant), PL14, PL17, and PL40. We revealed that PL7 belongs to subfamilies 3, 5, and 6. They may be involved in local and horizontal gene transfer and gene duplication processes. Most likely, an individual evolution of PL7 genes promotes the genetic variability of the Alginate Utilization System across Zobellia. Apparently, the PL7 alginate lyases may acquire a sub-functionalization due to diversification between in-paralogs.

Comparative Genomics and CAZyme Genome Repertoires of Marine Zobellia amurskyensis KMM 3526T and Zobellia laminariae KMM 3676T.

We obtained two novel draft genomes of type Zobellia strains with estimated genome sizes of 5.14 Mb for Z. amurskyensis KMM 3526Т and 5.16 Mb for Z. laminariae KMM 3676Т. Comparative genomic analysis has been carried out between obtained and known genomes of Zobellia representatives. The pan-genome of Zobellia genus is composed of 4853 orthologous clusters and the core genome was estimated at 2963 clusters. The genus CAZome was represented by 775 GHs classified into 62 families, 297 GTs of 16 families, 100 PLs of 13 families, 112 CEs of 13 families, 186 CBMs of 18 families and 42 AAs of six families. A closer inspection of the carbohydrate-active enzyme (CAZyme) genomic repertoires revealed members of new putative subfamilies of GH16 and GH117, which can be biotechnologically promising for production of oligosaccharides and rare monomers with different bioactivities. We analyzed AA3s, among them putative FAD-dependent glycoside oxidoreductases (FAD-GOs) being of particular interest as promising biocatalysts for glycoside deglycosylation in food and pharmaceutical industries.

Marine Bacterium Vibrio sp. CB1-14 Produces Guanidine Alkaloid 6-epi-Monanchorin, Previously Isolated from Marine Polychaete and Sponges.

Twenty-three bacterial strains were isolated from the secreted mucus trapping net of themarine polychaete Chaetopterus variopedatus (phylum Annelida) and twenty strains were identifiedusing 16S rRNA gene analysis. Strain CB1-14 was recognized as a new species of the genus Vibriousing the eight-gene multilocus sequence analysis (MLSA) and genome sequences of nineteen typeVibrio strains. This Vibrio sp. was cultured, and 6-epi-monanchorin (2), previously isolated from thepolychaete and two sponge species, was found in the cells and culture broth. The presence of the 6-epi-monanchorin was confirmed by its isolation followed by 1H NMR and HRESIMS analysis. Theseresults showed the microbial origin of the bicyclic guanidine alkaloid 2 in C. variopedatus.

Relationship between Adaptive Changing of Lysophosphatidylethanolamine Content in the Bacterial Envelope and Ampicillin Sensitivity of Yersinia pseudotuberculosis.

The low permeability of porin channels is the possible reason for Gram-negative bacterial resistance to antibiotics. The adaptive accumulation of lysophosphatidylethanolamine (LPE) in Yersinia pseudotuberculosis induces conformational changes of OmpF porin that may hinder the transport of antibiotics through this channel. The present study was aimed to test whether the changes in LPE content affect the resistance of bacteria to ampicillin. The addition of glucose to the culture medium was shown to simultaneously increase the level of LPE and minimum inhibitory concentration (MIC) for ampicillin of Y. pseudotuberculosis cells 6- and 2-fold, respectively. However, the coadministration of glucose and polyphenol extract from buckwheat husks reduced the content of LPE 2-fold and restored MIC to the control value. Thus, PBEH can be used as antibiotic adjuvant to improve an antibiotic's ability to cross the outer membrane. The present work demonstrated: (i) the role of adaptive changes in the lipid composition of Y. pseudotuberculosis in the development of antibiotic resistance, and (ii) the promising use of PBEH in combination therapy to increase the susceptibility of Gram-negative bacteria to the conventional ß-lactam antibiotics, probably attenuating in vivo a previously demonstrated effect of LPE on the conformation and function of the OmpF channel.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

BACKGROUND: Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES: The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS: The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS: The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION: The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC).

The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system.

Adaptive responses of outer membrane porin balance of Yersinia ruckeri under different incubation temperature, osmolarity, and oxygen availability.

The capability of Yersinia ruckeri to survive in the aquatic systems reflects its adaptation (most importantly through the alteration of membrane permeability) to the unfavorable environments. The nonspecific porins are a key factor contributing to the permeability. Here we studied the influence of the stimuli, such as temperature, osmolarity, and oxygen availability on regulation of Y. ruckeri porins. Using qRT-PCR and SDS-PAGE methods we found that major porins are tightly controlled by temperature. Hyperosmosis did not repress OmpF production. The limitation of oxygen availability led to decreased expression of both major porins and increased transcription of the minor porin OmpY. Regulation of the porin balance in Y. ruckeri, in spite of some similarities, diverges from that system in Escherichia coli. The changes in porin regulation can be adapted in Y. ruckeri in a species-specific manner determined by its aquatic habitats.