Structure and in vitro antiproliferative activity against breast cancer cells of the cell-wall polysaccharide from the marine bacterium Kangiella japonica KMM 3899T.

Kangiella japonica KMM 3899T is a Gram-negative bacterium isolated from a sandy sediment sample collected from the Sea of Japan. Here the results of the structure and the biological activity against breast cancer cells of the cell-wall polysaccharide from K. japonica KMM 3899T have been described. The structure of the repeating unit of the polysaccharide was elucidated using chemical analysis and NMR spectroscopy: →4)-α-L-GalpNAc3AcA-(1 â†’ 3)-α-D-GlcpNAc-(1 â†’ 4)-ß-D-GlcpNAc3NAcAN-(1→. The cell-wall polysaccharide had an antiproliferative effect against T-47D cells. Flow cytometric and Western blot analysis revealed that the polysaccharide induced S phase arrest and mitochondrial-dependent apoptosis.

Cell-cycle arrest and mitochondria-dependent apoptosis induction in T-47D cells by the capsular polysaccharide from the marine bacterium Kangiella japonica KMM 3897.

In this study, we reported the in vitro mechanisms of antiproliferative activity of capsular polysaccharide derived from marine Gram-negative bacteria Kangiella japonica KMM 3897 in human breast сarcinoma T-47D cells. Flow cytometric and Western blot analysis revealed that capsular polysaccharide effectively suppressed T-47D cell proliferation by inducing G0/G1 phase arrest and mitochondrial-dependent apoptosis. Moreover, polysaccharide influenced the ERK1/2 and p38 signaling pathways. The results of this study would enrich our understanding of the molecular mechanism of the anti-cancer activity of sulfated polysaccharides from marine Gram-negative bacteria.

Structure of the Lipooligosaccharide from the Deep-Sea Marine Bacterium Idiomarina zobellii KMM 231T, Isolated at a Depth of 4000 Meters.

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231T, isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain.

Streptocinnamides A and B, Depsipeptides from Streptomyces sp. KMM 9044.

The bacterium Streptomyces sp. KMM 9044 from a sample of marine sediment collected in the northwestern part of the Sea of Japan produces highly chlorinated depsiheptapeptides streptocinnamides A (1) and B (2), representatives of a new structural group of antibiotics. The structures of 1 and 2 were determined using nuclear magnetic resonance and mass spectrometry studies and confirmed by a series of chemical transformations. Streptocinnamide A potently inhibits Micrococcus sp. KMM 1467, Arthrobacter sp. ATCC 21022, and Mycobacterium smegmatis MC2 155.

Sulfated capsular polysaccharide from the marine bacterium Kangiella japonica inhibits T-47D cells growth in vitro.

Kangiella japonica KMM 3897 is a Gram-negative bacterium isolated from a coastal sea-water sample of the Sea of Japan. In this paper, the results about the structure and the antiproliferative effect on cancer cells of the capsular polysaccharide isolated from the Kangiella japonica KMM 3897 have been described. The carbohydrate polymer was isolated and purified by several separation techniques, and the structure was elucidated using chemical analysis and NMR spectroscopy. The following structure of the sulfated capsular polysaccharide, containing 2-amino-2-deoxy-D-mannuronic acid was established: The capsular polysaccharide exerted a selective antiproliferative effect and suppressed the colony formation of T-47D cells.

Structure of the Cell-Wall-Associated Polysaccharides from the Deep-Sea Marine Bacterium Devosia submarina KMM 9415T.

Two cell-wall-associated polysaccharides were isolated and purified from the deep-sea marine bacterium Devosia submarina KMM 9415T, purified by ultracentrifugation and enzymatic treatment, separated by chromatographic techniques, and studied by sugar analyses and NMR spectroscopy. The first polysaccharide with a molecular weight of about 20.7 kDa was found to contain d-arabinose, and the following structure of its disaccharide repeating unit was established: →2)-α-d-Araf-(1→5)-α-d-Araf-(1→. The second polysaccharide was shown to consist of d-galactose and a rare component of bacterial glycans-d-xylulose: →3)-α-d-Galp-(1→3)-ß-d-Xluf-(1→.

Harenicola maris gen. nov., sp. nov. isolated from the Sea of Japan shallow sediments.

A Gram-negative, non-motile bacterium КMM 3653T was isolated from a sediment sample from the Sea of Japan seashore, Russia. On the basis of the 16S rRNA gene sequence analysis the strain КMM 3653T was positioned within the family Rhodobacteraceae (class Alphaproteobacteria) forming a distinct lineage with the highest gene sequence similarities to the members of the genera Pacificibacter (95.2-94.7%) and Nioella (95.1-94.5%), respectively. According to the phylogenomic tree based on 400 conserved protein sequences, strain КMM 3653T was placed in the cluster comprising Vannielia litorea, Nioella nitratireducens, Litoreibacter albidus and Pseudoruegeria aquimaris as a separate lineage adjacent to V. litorea KCTC 32083T. The average nucleotide identity values between strain КMM 3653T and V. litorea KCTC 32083T, N. nitratireducens KCTC 32417T, L. albidus KMM 3851T, and P. aquimaris CECT 7680T were 71.1, 70.3, 69.6, and 71.0%, respectively. Strain КMM 3653T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid followed by C16:0. The polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid, two unidentified aminolipids, and five unidentified lipids. The DNA G+C content of 61.8% was calculated from the genome sequence. Based on the phylogenetic evidence and distinctive phenotypic characteristics, we proposed strain KMM 3653T (= KCTC 82575T) to be classified as a novel genus and species Harenicola maris gen. nov., sp. nov.

Structure and in vitro antiproliferative activity of the acidic capsular polysaccharide from the deep-sea bacterium Psychrobacter submarinus KMM 225T.

Psychrobacter submarinus KMM 225T is a Gram-negative bacterium isolated from a sea-water sample collected at a depth of 300 m in the Northwest Pacific Ocean. Here we report the structure of the capsular polysaccharide from P. submarinus KMM 225T and its effect on the viability and colony formation of cancer cells. The glycopolymer was purified by ultracentrifugation and chromatography methods, and the structure was elucidated using NMR spectroscopy and composition analyses. The following structure of the acidic capsular polysaccharide, containing 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-d-glucose [d-QuipNAc4N(S-Hb)] and 4,6-O-[(S)-1-carboxyethylidene]-2-acetamido-2-deoxy-d-glucose [d-GlcpNAc4,6(S-Pyr)] was established: The capsular polysaccharide slightly reduced the viability but effectively suppressed the colony formation of different types of cancer cells, of which the most pronounced inhibition was shown for the human chronic myelogenous leukemia K-562 cells.

Thalassobius aquimarinus sp. nov., isolated from the Sea of Japan seashore.

An aerobic, Gram-negative, non-pigmented non-motile bacterium designed КMM 8518T was isolated from a seawater sampled from the Sea of Japan seashore. Strain КMM 8518T grew at 7-42 °C and in the presence of 1-7% NaCl. The phylogenetic analyses based on 16S rRNA gene and whole-genome sequences placed the novel strain КMM 8518T into the genus Thalassobius as a separate lineage. Strain КMM 8518T shared the highest 16S rRNA gene sequence similarity of 98% to Thalassobius gelatinovorus KCTC 22092T and similarity values of ≤ 97% to other recognized Thalassobius species. The average nucleotide identity and digital DNA-DNA hybridization values between strain КMM 8518T and T. gelatinovorus KCTC 22092T were 79.6% and 23.5%, respectively. The major respiratory quinone was ubiquinone-10. The major fatty acid was C18:1ω7c followed by 11-methyl C18:1ω7c. Polar lipids comprised phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid, and three unidentified lipids. The DNA G+C content of 62.7% was calculated from genome sequence analysis. Based on the phylogenetic analyses and distinctive phenotypic characteristics, the marine bacterium КMM 8518T is concluded to represent a novel species of the genus Thalassobius for which the name Thalassobius aquimarinus sp. nov. is proposed. The type strain of the species is strain KMM 8518T (= KCTC 82576T).

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

Structure and in vitro Bioactivity against Cancer Cells of the Capsular Polysaccharide from the Marine Bacterium Psychrobacter marincola.

Psychrobacter marincola KMM 277T is a psychrophilic Gram-negative bacterium that has been isolated from the internal tissues of an ascidian Polysyncraton sp. Here, we report the structure of the capsular polysaccharide from P. marincola KMM 277T and its effect on the viability and colony formation of human acute promyelocytic leukemia HL-60 cells. The polymer was purified by several separation methods, including ultracentrifugation and chromatographic procedures, and the structure was elucidated by means of chemical analysis, 1-D, and 2-D NMR spectroscopy techniques. It was found that the polysaccharide consists of branched hexasaccharide repeating units containing two 2-N-acetyl-2-deoxy-d-galacturonic acids, and one of each of 2-N-acetyl-2-deoxy-d-glucose, d-glucose, d-ribose, and 7-N-acetylamino-3,5,7,9-tetradeoxy-5-N-[(R)-2-hydroxypropanoylamino]- l-glycero-l-manno-non-2-ulosonic acid. To our knowledge, this is the first finding a pseudaminic acid decorated with lactic acid residue in polysaccharides. The biological analysis showed that the capsular polysaccharide significantly reduced the viability and colony formation of HL-60 cells. Taken together, our data indicate that the capsular polysaccharide from P. marincola KMM 277T is a promising substance for the study of its antitumor properties and the mechanism of action in the future.

Structure of phosphorylated and sulfated polysaccharides from lipopolysaccharide of marine bacterium Marinicella litoralis KMM 3900T.

Two polysaccharide fractions were obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Marinicella litoralis KMM 3900T. The major polysaccharide was found to contain glycerol 1-phosphate (PGro) and methyl phosphate substituents (PMe), and the following structure of its disaccharide repeating unit was established by sugar analysis, dephosphorylation, Smith degradation, and 1D and 2D NMR spectroscopy: →4)-α-L-Rhap2PGro(~40%)-(1 â†’ 3)-ß-D-Manp6PMe(~80%)-(1 â†’ . The minor polysaccharide was shown to consist of 4-O-sulfate-d-mannopyranosyl residues, non-stoichiometric methylated at O-3 and acetylated at O-6: →2)-α-D-Manp3R4S6Ac(~75%)-(1→, where R is Me (85%) or H (15%).

Structure and bioactivity of sulfated α-D-mannan from marine bacterium Halomonas halocynthiae KMM 1376T.

Halomonas halocynthiae KMM 1376T is a Gram-negative bacterium that has been isolated from gill tissue of the ascidian Halocynthia aurantium. Mild acid hydrolysis of the lipopolysaccharide of H. halocynthiae KMM 1376T afforded an O-polysaccharide, which was studied by sugar analysis and NMR spectroscopy. The following structure of the O-polysaccharide presented as sulfated α-D-mannan was established: →2)-α-D-Manp3,6S-(1→3)-α-D-Manp2Ac(∼71%)6S-(1→3)-α-D-Manp-(1→. Study of biological activity has shown that sulfated α-D-mannan can specifically reduce the cell viability and colony formation of the human breast adenocarcinoma MDA-MB-231 cells in a concentration-dependent manner. In addition, polysaccharide inhibits epidermal growth factor induced neoplastic cell transformation in mouse epidermal JB6 Cl41 cells.

Structure, antiproliferative and cancer preventive properties of sulfated α-d-fucan from the marine bacterium Vadicella arenosi.

Sulfated fucose-containing glycopolymers are currently of great interest because of their wide spectrum of bioactivity, including anti-tumor properties. In this study, the structure of O-polysaccharide (OPS) of the marine bacterium Vadicella arenosi KMM 9024T, its effect on the proliferation of human breast cancer MCF-7 cells and cancer preventive properties were investigated. Two OPS fractions with different molecular weights were isolated and purified from the lipopolysaccharide by mild acid hydrolysis followed by anion-exchange chromatography. The OPS was found to consist of α-(1→3)-linked 2-O-sulfate-d-fucopyranosyl residues, whose structure was deduced by sugar analysis along with 2D NMR spectroscopy. The biological assay indicated that polysaccharide significantly reduced the proliferation and inhibited colony formation of MCF-7 cells in a dose-dependent manner. Besides, the experiment indicated the inhibitory role of polysaccharide on EGF-induced neoplastic cell transformation in mouse epidermal cells. The investigated polysaccharide is the first sulfated fucan isolated from the bacteria.

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).

Winogradskyella algae sp. nov., a marine bacterium isolated from the brown alga.

An aerobic, Gram-negative, yellow-pigmented non-motile rod-shaped bacterium Kr9-9T was isolated from a brown alga specimen collected near the Kuril Islands. Based on the 16S rRNA gene sequence analysis strain Kr9-9T was assigned to the genus Winogradskyella, and its close phylogenetic neighbors were found to be Winogradskyella damuponensis KCTC 23552T, Winogradskyella sediminis LMG 28075T, and Winogradskyella rapida CCUG 59098T showing high similarities of 98.1%, 97.5%, and 97.1%, respectively. It contained MK-6 as the predominant menaquinone and iso-C15:0, anteiso-C15:0, iso-C16:0 3-OH followed by iso-C15:1 as the major fatty acids. Polar lipids included phosphatidylethanolamine, three unidentified aminolipids and an unidentified lipid. The DNA C+C content was 32.3 mol%. Based on the phylogenetic analysis and distinctive phenotypic characteristics, strain Kr9-9T is concluded to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella algae sp. nov. is proposed. The type strain of the species is strain Kr9-9T (= KMM 8180T = KACC 19709T).

Sulfated O-polysaccharide with anticancer activity from the marine bacterium Poseidonocella sedimentorum KMM 9023T.

The sulfated polysaccharides are of study interest due to their high structural diversity and broad spectrum of biological activity including antitumor properties. In this paper, we report on the structural analysis of sulfated O-specific polysaccharide (OPS) and in vitro anticancer activity of O-deacylated lipopolysaccharide (DPS) of the marine-derived bacterium Poseidonocella sedimentorum KMM 9023T achieved by a multidisciplinary approach (chemical analysis, NMR, MS, and bioassay). The OPS is shown to include two rare monosaccharide derivatives: 3-deoxy-9-O-methyl-d-glycero-d-galacto-non-2-ulosonic acid (Kdn9Me) and 3-O-acetyl-2-O-sulfate-d-glucuronic acid (D-GlcA2S3Ac). The structure of polysaccharide moiety of a previously unknown carbohydrate-containing biopolymer is established: →4)-α-Kdnp9Me-(2→4)-α-d-GlcpA2S3Ac-(1→. From a biological point of view, we demonstrate that DPS of the P. sedimentorum KMM 9023T has no cytotoxicity and inhibits colony formation of human HT-29, MCF-7 and SK-MEL-5 cells in a dose-dependent manner. The investigated polysaccharide is the second glycan isolated from the bacteria of the genus Poseidonocella: previously we studied the OPS of P. pacifica KMM 9010T (Kokoulin et al., 2017). Both polysaccharides are sulfated and contain rare residues of ulosonic acids. Thus, obtained findings provide a new knowledge about kinds and antitumor properties of sulfated polysaccharides and can be a starting point for further investigations of mechanisms of anticancer action of carbohydrate-containing biopolymers from marine Gram-negative bacteria.

5-Acetamido-3,5-dideoxy-L-glycero-L-manno-non-2-ulosonic acid-containing O-polysaccharide from marine bacterium Pseudomonas glareae KMM 9500T.

The O-polysaccharide was isolated from the lipopolysaccharide of a marine bacterium Pseudomonas glareae KMM 9500T and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy including 1H,1H-TOCSY, 1H,1H-COSY, 1H,1H-ROESY, 1H,13C-HSQC and 1H,13C-HMBC experiments. The O-polysaccharide was found to consist of linear tetrasaccharide repeating units constituted by D-glucuronic acid (D-GlcA), L-rhamnose (L-Rha), D-glucose (D-Glc) and 5-acetamido-7,9-O-[(S)-1-carboxyethylidene]-3,5-dideoxy-L-glycero-L-manno-non-2-ulosonic acid (Sug7,9(S-Pyr)), partially O-acetylated at position 8 (∼70%): →4)-α-D-GlcpA-(1→3)-ß-L-Rhap-(1→4)-ß-D-Glcp-(1→4)-ß-Sugp8Ac(∼70%)7,9(S-Pyr)-(2→.

Structure of 3,6-dideoxy-3-[(R)-2-hydroxypropanoylamino]-D-galactose-containing O-polysaccharide from marine bacterium Simiduia litorea KMM 9504T.

The O-polysaccharide was isolated from the lipopolysaccharide of a marine bacterium Simiduia litorea KMM 9504T and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy including 1H,1H-TOCSY, 1H,1H-COSY, 1H,1H-ROESY, 1H,13C-HSQC and 1H,13C-HMBC experiments. The following new structure of the O-polysaccharide of S. litorea KMM 9504T containing D-galacturonamide, 2-acetamido-2,6-dideoxy-D-glucose and 3,6-dideoxy-3-[(R)-2-hydroxypropanoylamino]-D-galactose was established.