Genome-Based Classification of Strain 16-SW-7, a Marine Bacterium Capable of Converting B Red Blood Cells, as Pseudoalteromonas distincta and Proposal to Reclassify Pseudoalteromonas paragorgicola as a Later Heterotypic Synonym of Pseudoalteromonas distincta.

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain 16-SW-7, isolated from a seawater sample, was investigated in detail due to its ability to produce a unique α-galactosidase converting B red blood cells into the universal type blood cells. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain 16-SW-7 is a member of the Gammaproteobacteria genus Pseudoalteromonas. The closest relatives of the environmental isolate were Pseudoalteromonas distincta KMM 638T and Pseudoalteromonas paragorgicola KMM 3548T, with the plural paralogous 16S rRNA genes of 99.87-100% similarity. The strain 16-SW-7 grew with 1-10% NaCl and at 4-34°C, and hydrolyzed casein, gelatin, tyrosine, and DNA. The genomic DNA G+C content was 39.3 mol%. The prevalent fatty acids were C16:1 ω7c, C16:0, C17:1 ω8c, C18:1 ω7c, C17:0, and C12:0 3-OH. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, two unidentified amino lipids, and three unidentified lipids. The major respiratory quinone was Q-8. The finished genome of the strain 16-SW-7 (GenBank assembly accession number: GCA_005877035.1) has a size of 4,531,445 bp and comprises two circular chromosomes L1 and S1, deposited in the GenBank under the accession numbers CP040558 and CP040559, respectively. The strain 16-SW-7 has the ANI values of 98.2% with KMM 638T and KMM 3548T and the DDH values of 84.4 and 83.5%, respectively, indicating clearly that the three strains belonged to a single species. According to phylogenetic evidence and similarity for the chemotaxonomic and genotypic properties, the strain 16-SW-7 (= KCTC 52772 = KMM 701) represents a novel member of the species Pseudoalteromonas distincta. Also, we have proposed to reclassify Pseudoalteromonas paragorgicola as a later heterotypic synonym of P. distincta based on the rules of priority with the emendation of the species.

Production of Vitamin B2 (Riboflavin) by Microorganisms: An Overview.

Riboflavin is a crucial micronutrient that is a precursor to coenzymes flavin mononucleotide and flavin adenine dinucleotide, and it is required for biochemical reactions in all living cells. For decades, one of the most important applications of riboflavin has been its global use as an animal and human nutritional supplement. Being well-informed of the latest research on riboflavin production via the fermentation process is necessary for the development of new and improved microbial strains using biotechnology and metabolic engineering techniques to increase vitamin B2 yield. In this review, we describe well-known industrial microbial producers, namely, Ashbya gossypii, Bacillus subtilis, and Candida spp. and summarize their biosynthetic pathway optimizations through genetic and metabolic engineering, combined with random chemical mutagenesis and rational medium components to increase riboflavin production.

Development of host strains and vector system for an efficient genetic transformation of filamentous fungi.

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 µg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with ß-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

Polaribacter staleyi sp. nov., a polysaccharide-degrading marine bacterium isolated from the red alga Ahnfeltia tobuchiensis.

A Gram-stain-negative, rod-shaped, motile by gliding and yellow-pigmented bacterium, designated strain 10Alg 139T, was isolated from the Pacific red alga Ahnfeltiato buchiensis. The phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain belonged to the genus Polaribacter, a member of the family Flavobacteriaceae, the phylum Bacteroidetes, with highest sequence similarity to Polaribacter butkevichii KMM 3938T (99.3 %) and 93.3-98.6 % to other recognized Polaribacter species. The prevalent fatty acids of strain 10Alg 139T were iso-C15 : 0 3-OH, C15 : 0 3-OH, iso-C15:0, iso-C13 : 0, C15 : 0 and C15 : 1ω6c. The polar lipid profile consisted of the major lipids phosphatidylethanolamine, two unidentified aminolipids and four unidentified lipids. The main respiratory quinone was menaquinone 6. The DNA G+C content of the type strain is 31.8 mol%. The new isolate and the type strains of recognized species of the genus Polaribacter were readily distinguished based on a number of phenotypic characteristics. A combination of the genotypic and phenotypic data showed that the isolate from alga represents a novel species of the genus Polaribacter, for which the name Polaribacterstaleyi sp. nov. is proposed. The type strain is 10Alg 139T (=KCTC 52773T=KMM 6729T).

Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis.

α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.

Carbohydrate-binding motifs in a novel type lectin from the sea mussel Crenomytilus grayanus: Homology modeling study and site-specific mutagenesis.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL-GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1-3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL-GalNAc binding energies. We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.

Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas.

The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the Escherichia coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy. The kinetics of formation of α- and ß-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of ß-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

Flavobacterium ahnfeltiae sp. nov., a new marine polysaccharide-degrading bacterium isolated from a Pacific red alga.

A Gram-negative, aerobic, rod-shaped, motile by gliding and yellow-pigmented bacterium, designated strain 10Alg 130(T), that displayed the ability to destroy polysaccharides of red and brown algae, was isolated from the red alga Ahnfeltia tobuchiensis. The phylogenetic analysis based on 16S rRNA gene sequence placed the novel strain within the genus Flavobacterium, the type genus of the family Flavobacteriaceae, the phylum Bacteroidetes, with sequence similarities of 96.2 and 95.7 % to Flavobacterium jumunjiense KCTC 23618(T) and Flavobacterium ponti CCUG 58402(T), and 95.3-92.5 % to other recognized Flavobacterium species. The prevalent fatty acids of strain 10Alg 130(T) were iso-C15:0, iso-C15:0 3-OH, iso-C17:0 3-OH, C15:0 and iso-C17:1ω9c. The polar lipid profile consisted of phosphatidylethanolamine, two unknown aminolipids and three unknown lipids. The DNA G+C content of the type strain was 34.3 mol%. The new isolate and the type strains of recognized species of the genus Flavobacterium could strongly be distinguished by a number of phenotypic characteristics. A combination of the genotypic and phenotypic data showed that the algal isolate represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ahnfeltiae sp. nov. is proposed. The type strain is 10Alg 130(T) (=KCTC 32467(T) = KMM 6686(T)).

An extracellular S1-type nuclease of marine fungus Penicillium melinii.

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5-8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3'-5' mode to produce 5'-oligonucleotides and 5'-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 alpha-galactosidase.

An alpha-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na+ ions and temperature up to 34 degrees C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular alpha-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The alpha-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric alpha-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This alpha-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.

Effect of 1,3;1,6-beta-D-glucans on developing sea urchin embryos.

The effect of 1,3;1,6-beta-D-glucooligo- and polysaccharides with different structures (from 1 to 10 kDa of molecular mass; from 10-25% of beta-1,6-linked glucose residues content) on the developing embryos of sea urchin, Strongylocentrotus intermedius, was evaluated for the screening of potential positive stimulants. 1,3;1,6-beta-D-glucans with a molecular mass of between 6-10 kDa and at concentrations of 0.05-0.25 mg/ml shown the best modulator effect on the sea urchin embryos. 1,3;1,6-beta-D-glucans increased the survival of the sea urchin embryos up to 2.5-fold compared with the control animals.