Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

Aquimarina algiphila sp. nov., a chitin degrading bacterium isolated from the red alga Tichocarpus crinitus.

A strictly aerobic, Gram-stain-negative, rod-shaped, motile by gliding and yellow-orange pigmented flavobacterium, designated strain 9Alg 151T, was isolated from the Pacific red alga Tichocarpus crinitus. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain fell into the genus Aquimarina of the family Flavobacteriaceae with a 16S rRNA gene sequence similarity range of 94.2-98.2 % to the recognized species of the genus. Strain 9Alg 151T grew in the presence of 0.5-5 % NaCl and at 5-34 °C, and hydrolysed aesculin, agar, gelatin, starch, Tween 40, DNA and chitin. The predominant fatty acids were iso-C17 : 0 3-OH, iso-C15 : 0, iso-C15 : 1 G, iso-C15 : 0 3-OH, iso-C16 : 0, iso-C17 : 1ω8c and summed feature 3. The polar lipid profile comprised phosphatidylethanolamine, three unidentified aminolipids and three unidentified lipids. The major respiratory quinone was MK-6. The genomic DNA G+C content was 32.6 mol%. On the basis of 16S rRNA gene sequence data, and chemotaxonomic and phenotypic characteristics, strain 9Alg 151T represents a novel species of the genus Aquimarina, for which the name Aquimarina algiphila sp. nov. is proposed. The type strain is 9Alg 151T (=KCTC 23622T=KMM 6462T).

Echinimonas agarilytica gen. nov., sp. nov., a new gammaproteobacterium isolated from the sea urchin Strongylocentrotus intermedius.

A novel Gram-negative, facultatively anaerobic and motile bacterial strain, designated KMM 6351(T), was isolated from the sea urchin Strongylocentrotus intermedius and examined using a polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a distinct phyletic line in the class Gammaproteobacteria and was most closely related to the genera Aliivibrio, Photobacterium and Vibrio. Strain KMM 6351(T) grows at 4-40 °C and with 0.5-12 % NaCl and decomposes aesculin, agar, gelatin, starch, chitin and DNA. The DNA G+C content of the strain was determined to be 46.1 mol%. The prevalent fatty acids were found to be C(16:0), C(18:1) ω7c, C(12:0) 3-OH and summed feature 3 (comprising C(16:1) ω7c and/or iso-C(15:0) 2-OH fatty acids). The major polar lipids were determined to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The predominant ubiquinone was found to be Q-8. The results of the phenotypic, chemotaxonomic and genotypic analyses clearly indicated that the novel strain should be assigned to a new genus and species within the class γ-Proteobacteria for which the name Echinimonas agarilytica gen. nov., sp. nov. is proposed. The type strain is KMM 6351(T) (=KCTC 22996(T) = LMG 25420(T)).

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies.

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the ß-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.

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.

Trends of the major porin gene (ompF) evolution: insight from the genus Yersinia.

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species.