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.

A new multigene HCIQ subfamily from the sea anemone Heteractis crispa encodes Kunitz-peptides exhibiting neuroprotective activity against 6-hydroxydopamine.

The Kunitz/BPTI-type peptides are ubiquitous in numerous organisms including marine venomous animals. The peptides demonstrate various biological activities and therefore they are the subject of a number of investigations. We have discovered a new HCIQ subfamily belonging to recently described multigene HCGS family of Heteractis crispa Kunitz-peptides. The uniqueness of this subfamily is that the HCIQ precursors contain a propeptide terminating in Lys-Arg (endopeptidase cleavage site) the same as in the neuro- and cytotoxin ones. Moreover, the HCIQ genes contain two introns in contrast to HCGS genes with one intron. As a result of Sanger and amplicon deep sequencings, 24 HCIQ isoforms were revealed. The recombinant peptides for the most prevalent isoform (HCIQ2c1) and for the isoform with the rare substitution Gly17Glu (HCIQ4c7) were obtained. They can inhibit trypsin with Ki 5.2 × 10-8 M and Ki 1.9 × 10-7 M, respectively, and interact with some serine proteinases including inflammatory ones according to the SPR method. For the first time, Kunitz-peptides have shown to significantly increase neuroblastoma cell viability in an in vitro 6-OHDA-induced neurotoxicity model being a consequence of an effective decrease of ROS level in the cells.

Characterization of Labrenzia polysiphoniae sp. nov. isolated from red alga Polysiphonia sp.

A group of five Gram-negative aerobic halophilic bacteria was isolated from the red alga Polysiphonia sp. specimen collected from the Sea of Japan seashore and subjected to a taxonomic study. On the basis of 16S rRNA gene sequence analysis, the novel isolates were affiliated to the genus Labrenzia sharing the highest gene sequence similarities of 98.1-98.4% with the type strain of Labrenzia suaedae KACC 13772T. The DNA-DNA hybridization values of 83-91% obtained between five novel strains, and 26 and 36% between two of the five novel strains and the closest neighbor Labrenzia suaedae KACC 13772T confirmed their assignment to the same separate species. Novel isolates were characterized by Q-10 as the major ubiquinone, by the predominance of C18:1ω7c followed by 11-methyl C18:1ω7c and C14:0 3-ОН in their fatty acid profiles. Polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, an unknown aminophospholipid, and an unknown phospholipid. Some of novel strains were found to inhibit growth of Gram-negative and Gram-positive test microorganisms. On the basis of phylogenetic analysis, DNA-DNA hybridization and phenotypic traits, a novel species with the name Labrenzia polysiphoniae sp. nov. (type strain KMM 9699T = rh46T = KACC 19711T), is proposed.

Winogradskyella profunda sp. nov. isolated from the Chukchi Sea bottom sediments.

An aerobic, Gram-negative, yellow-pigmented non-motile rod-shaped bacterium, designated Ch38T, was isolated from a sediment sample collected from the Chukchi Sea in the Arctic Ocean. Comparative 16S rRNA gene sequence analysis positioned strain Ch38T into the genus Winogradskyella as a distinct line adjacent to Winogradskyella multivorans KCTC 23891T, sharing the highest similarities of 97.5%, 97.2%, and 97.1% with Winogradskyella eximia KCTC 12219T, Winogradskyella damuponensis KCTC 23552T, and Winogradskyella multivorans KCTC 23891T, respectively. Strain Ch38T grew at 5-36 °C and in the presence of 1-6% (w/v) NaCl. It contained MK-6 as the predominant menaquinone and iso-C16:0 3-OH, anteiso-C15:0 followed by iso-C15:0 and iso-C16:1 as the major fatty acids. Polar lipids consisted of phosphatidylethanolamine, three unknown aminolipids, an unknown lipid and an unknown phospholipid. The DNA C + C content was 31.7 mol%. Based on the phylogenetic analysis and distinctive phenotypic characteristics, strain Ch38T is concluded to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella profunda sp. nov. is proposed. The type strain of the species is strain Ch38T (= KMM 9725T = KACC 19710T).

Multigene Family of Pore-Forming Toxins from Sea Anemone Heteractis crispa.

Sea anemones produce pore-forming toxins, actinoporins, which are interesting as tools for cytoplasmic membranes study, as well as being potential therapeutic agents for cancer therapy. This investigation is devoted to structural and functional study of the Heteractis crispa actinoporins diversity. Here, we described a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. The phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families. The transcriptomes of both H. crispa and Heteractis magnifica appear to contain a large repertoire of similar genes representing a rapid expansion of the actinoporin family due to gene duplication and sequence divergence. The presence of the most abundant specific group of actinoporins in H. crispa is the major difference between these species. The functional analysis of six recombinant actinoporins revealed that H. crispa actinoporin grouping was consistent with the different hemolytic activity of their representatives. According to molecular modeling data, we assume that the direction of the N-terminal dipole moment tightly reflects the actinoporins' ability to possess hemolytic activity.

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.

A new multigene superfamily of Kunitz-type protease inhibitors from sea anemone Heteractis crispa.

Despite a considerable number of publications devoted to isolation and physicochemical properties of protease inhibitors from sea anemones, virtually nothing is known about the structure of the genes, and the nature of their isoforms diversity. Using the PCR-based cloning approach we discovered the Kunitz-type multigene superfamily composed of distinct gene families (GS-, RG-, GG-, and GN-gene families). It has been identified only three full-length GS-transcripts indicating a much greater variety of Kunitz homologs in Heteractis crispa. We have examined an exon-intron structure of GS-genes; an open reading frame is interrupted by a single intron located at the middle of the signal peptide. 33 deduced mature GS-polypeptides have been categorized into three groups according to the nature of a P1 residue. Some of them corresponded to native Kunitz-type protease inhibitors earlier isolated from H. crispa. The deduced GS-polypeptide sequences demonstrated diverse charge distribution ranging from the local point charges forms to the overall positive ones. We have suggested that the GS-gene family has evolved through gene tandem duplication followed by adaptive divergence of the P1 residue in the reactive site selected for divergent functions in paralogs. The expansion of this Kunitz-type multigene superfamily during evolution is lineage-specific, providing the tropical sea anemone H. crispa with the ability to interact an increasing diversity of the preys and predators. Our results show that the Kunitz-type polypeptides are encoded by a multigene superfamily and realized via a combinatory Kunitz-type library in the H. crispa tentacles venom.

An endo-(1-->3)-beta-D-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization.

An endo-(1-->3)-beta-d-glucanase (L(0)) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1-->3)-beta-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 degrees C. L(0) hydrolyzed laminaran with K(m) approximately 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl betad-glucoside as acceptor (K(m) approximately 2mg/mL for laminaran) and laminaran as donor and as acceptor (K(m) approximately 5mg/mL) yielding p-nitrophenyl betad-glucooligosaccharides (n=2-6) and high-molecular branching (1-->3),(1-->6)-beta-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of beta-(1-->6)-glycosidic bonds, and laminaran with 10% of beta-(1-->6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L(0) was characteristic for a protein with prevailing beta secondary-structural elements. Binding L(0) with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L(0) with glucose (K(a)=7.4 x 10(5)+/-1.1 x 10(5)M(-1)) and stoichiometry (n=13.3+/-0.7) was calculated. The cDNA encoding L(0) was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.

Amino acid sequence of RTX-A's isoform actinoporin from the sea anemone, Radianthus macrodactylus.

The amino acid sequence of actinoporin RTX-A (175 aa) from the sea anemone Radianthus macrodactylus was determined by sequencing of clones obtained via amplification of cDNA. It was established that RTX-A possessed high homology with HmgIII from Heteractis magnifica (87%) and StI, StII from Stichodactyla helianthus (84 and 87%, respectively). The analysis of structural and functional relationships within RTX-A was carried out. The some disagreement concerning to significant role of several amino acid residues for actinoporins exhibition of hemolytic activity was found.

Purification, cDNA cloning and homology modeling of endo-1,3-beta-D-glucanase from scallop Mizuhopecten yessoensis.

The retaining endo-1,3-beta-D-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products (Km approximately 600 microg/mL). The 1,3-beta-D-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 degrees C, respectively. cDNA encoding the endo-1,3-beta-D-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-beta-D-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-beta-D-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and beta-1,3-glucan-binding proteins. The fold of the LV was more closely related to kappa-carrageenase, agarase and 1,3;1,4-beta-D-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-beta-D-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of kappa-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.

Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus.

A new cytolytic toxin, actinoporin RTX-S II, was isolated from the sea anemone Radianthus macrodactylus with a high degree of purity by a combination of gel filtration, ion-exchange and reverse-phase chromatography. RTX-S II has molecular mass of 19,280 Da and isoelectric point of 10.0. The hemolytic activity of RTX-S II is inhibited by sphingomyelin. RTX-S II had an LD(50) of 70 mg/kg, and is lacking in phospholipase activity. The amino acid composition of this protein contains a high amount of basic and non-polar amino acids and no cysteine. The N-terminal sequence of RTX-S II was determined. The partial amino acid sequence (141 aa) of RTX-S II was deduced based on the cDNA sequence obtained with two oligonucleotides encoding the N-terminal portion of RTX-S II and the internal conserved cytolysin peptide by PCR. A comparison of the RTX-S II cDNA sequence and the rtx-s II gene obtained with the same PCR primers indicates that they are 100% identical at the nucleotide level. It shows that no introns are present in the corresponding region of the rtx-s II gene. Multiple alignments of RTX-S II with known sequences of actinoporins show that RTX-S II is highly homologous to magnificalysin II from Heteractis magnifica. The predicted secondary structure of RTX-S II is predominantly anti-parallel beta-structure, which is in good agreement with experimental data obtained from other sea anemones-actinoporins.