Valeriya V. Kurilenko

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium, Formosa algae

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg2+, Ca2+ and Ba2+ cations strongly activated the enzyme; however, Cu2+ and Zn2+ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.

Winogradskyella exilis sp. nov., isolated from the starfish Stellaster equestris, and emended description of the genus Winogradskyella

A pale-yellowish-pigmented strain, 022-2-26(T), was isolated from a starfish, Stellaster equestris. Cells of strain 022-2-26(T) were Gram-negative short rods that were chemo-organotrophic, alkalitolerant and mesophilic. The predominant menaquinone was MK-6. The major cellular fatty acids were iso-C(15 : 0), iso-C(15 : 1), C(15 : 0), iso-C(15 : 0) 2-OH and iso-C(17 : 0) 3-OH (together representing 87 % of the total fatty acids). The DNA G+C content was 30.1 mol%. A 16S rRNA gene sequence of the isolate was determined and phylogenetic analyses revealed that strain 022-2-26(T) formed a robust clade (neighbour-joining algorithm with a bootstrap value of 95 % and parsimony and maximum-likelihood algorithms) with type strains of species in the genus Winogradskyella. The closest phylogenetic neighbour of strain 022-2-26(T) was Winogradskyella poriferorum UST030701-295(T) (96 % 16S rRNA gene sequence similarity; 59 differences between sequences). On the basis of the phenotypic and chemotaxonomic characteristics and the phylogenetic evidence, it is proposed that strain 022-2-26(T) represents a novel species, Winogradskyella exilis sp. nov. The type strain is 022-2-26(T) (=KMM 6013(T) =CIP 109976(T)).

Luteimonas vadosa sp. nov., isolated from seashore sediment.

An aerobic, Gram-stain-negative, yellow-pigmented, non-motile, rod-shaped bacterium designated strain KMM 9005(T) was isolated from a sediment sample collected from the Sea of Japan seashore. Comparative 16S rRNA gene sequence analysis showed that strain KMM 9005(T) belonged to the genus Luteimonas and was most closely related to Luteimonas cucumeris KCTC 23627(T) (96.5 % sequence similarity) and Luteimonas aquatica LMG 24212(T) (96.1 % sequence similarity). Strain KMM 9005(T) was characterized by the presence of thin fimbriae, the major ubiquinone Q-8, by the predominance of iso-C17 : 1 followed by iso-C16 : 0, iso-C15 : 0 and iso-C17 : 0 in its fatty acid profile, weak hydrolytic capacity and the inability to assimilate most organic substrates. Based on these distinctive phenotypic characteristics and phylogenetic analysis, strain KMM 9005(T) represents a novel species of the genus Luteimonas, for which the name Luteimonas vadosa sp. nov. is proposed. The type strain is KMM 9005(T) ( = NRIC 0881(T) = JCM 18392(T)).

Celeribacter neptunius gen. nov., sp. nov., a new member of the class Alphaproteobacteria

A whitish Gram-negative, motile, aerobic bacterium, designated strain H 14(T), was isolated from seawater collected at St Kilda beach in Port Phillip Bay, Melbourne, Australia. Analysis of 16S rRNA gene sequences revealed that the organism belonged to the Roseobacter lineage of the class Alphaproteobacteria, forming a distinct evolutionary lineage at the genus level. Strain H 14(T) was distantly related to the genera Nautella, Ruegeria and Pseudoruegeria (family Rhodobacteraceae). Strain H 14(T) was unable to degrade gelatin, casein, chitin, agar and starch, did not produce any carotenoids, did not possess bacteriochlorophyll a and had a limited ability to utilize carbon sources. Strain H 14(T) grew with concentrations of 1-8 % (w/v) NaCl and over a temperature range of 5-35 degrees C. Phosphatidylglycerol was the major phospholipid (90 %); phosphatidylcholine (7.9 %) and phosphatidylethanolamine (2.0 %) were present in minor quantities. The predominant fatty acids were C(18 : 1)omega7c (82.4 %), C(18 : 1)omega9c (5.1 %) and C(18 : 0) (3.8 %). The DNA G+C composition for strain H 14(T) was 59.1 mol%. Based on the results of physiological, biochemical, chemotaxonomic and phylogenetic investigations, a new genus, Celeribacter gen. nov., with the type species Celeribacter neptunius sp. nov. is proposed. The type strain of the type species is H 14(T) (=KMM 6012(T)=CIP 109922(T)).

Loktanella maritima sp. nov. isolated from shallow marine sediments.

An aerobic, Gram-stain-negative, non-motile bacterium, KMM 9530(T), was isolated from a sediment sample collected from the Sea of Japan seashore. Comparative 16S rRNA gene sequence analysis positioned novel strain KMM 9530(T) in the genus Loktanella as a separate line adjacent to Loktanella sediminilitoris KCTC 32383(T), Loktanella tamlensis JCM 14020(T) and Loktanella maricola JCM 14564(T) with 98.5-98.2% sequence similarity. Strain KMM 9530(T) was characterized by its weak hydrolytic capacity and inability to assimilate most organic substrates. The major isoprenoid quinone was Q-10, polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid, an unknown aminolipid and unknown lipids, and the major fatty acid was C18 : 1ω7c. On the basis of phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization, it can be concluded that the novel strain KMM 9530(T) represents a novel species in the genus Loktanella, for which the name Loktanella maritima sp. nov. is proposed. The type strain of the species is KMM 9530(T) ( = NRIC 0919(T) = JCM 19807(T)).

Zonal distribution of epiphytic microorganisms on the eelgrass Zostera marina

The structure and the functioning of marine bacterial communities colonizing submarine perennial grasses of the family Zosteraceae are largely determined by the complex trophic interrelations between the grasses and their epiphytes. The productivity and the biomass of seaweeds, some species of which are used as food or the sources of valuable biologically active compounds [1], are regulated by various biotic and abiotic factors [2]. Seaweeds are colonized by epiphytes, which are represented by microalgae, bacteria, microscopic fungi, and protozoans. When the population of epiphytic organisms appears too large for the seaweed leaves to obtain sufficient light, there may be disturbances in their carbon metabolism and a substantial decrease in the productivity of the marine communities [3, 4]. A symbiotrophic relationship between epiphytic bacteria and the eelgrass Zostera marina allows these bacteria to take organic carbon, phosphorus, and nitrogen from the eelgrass leaves [2, 5]. It should be noted that the investigations of the taxonomic diversity of epiphytic bacteria on Zostera marina are scarce and limited, to the best of our knowledge, to two studies reporting the isolation of bacteria of the genera Flavobacterium, Pseudomonas , and Vibrio from the surface of this eelgrass [6, 7].

Phospholipids of marine proteobacteria of the genusPseudoalteromonas

The study of the phospholipid composition of 14 type strains of marine proteobacteria of the genusPseudoalteromonas showed that phospholipids are the main polar lipid constituents of membranes in these proteobacteria. The phospholipid patterns of the strains studied were found to be similar and involved five phospholipids typical of gram-negative bacteria, namely, phosphatidylethanolamine, phosphatidylglycerol, bisphosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid. The major phospholipids were phosphatidylethanolamine and phosphatidylglycerol, which add up to 89–97% of the total phospholipids; bisphosphatidic acid was dominant among minor phospholipids. The prevalence of phosphatidylethanolamine (62–77% of the total phospholipids) and the absence of diphosphatidylglycerol are the characteristic features of most bacteria of this genus. As inEscherichia coli, the phospholipid composition of the marine proteobacteria depended on the presence of magnesium in the medium.

Tamlana sedimentorum sp. nov., isolated from shallow sand sediments of the Sea of Japan.

An aerobic, Gram-stain-negative, saffron-pigmented, non-motile bacterial strain KMM 9545(T), was isolated from a marine sand sediment sample obtained from the Sea of Japan seashore and characterized in a taxonomic study using a polyphasic approach. The 16S rRNA gene sequence of strain KMM 9545(T) showed a high level of similarity to species of the genus Gaetbulibacter (95.1-96.2%), the type strains of species of the genus Tamlana (94.9-96.1%) and members of the genus Algibacter (94.8-96.1%). Phylogenetic analysis based on the 16S rRNA gene sequences positioned strain KMM 9545(T) as a distinct lineage in the cluster comprising species of the genus Tamlana. Strain KMM 9545(T) grew at temperatures between 5-36 °C and in the presence of 2-4% (w/v) NaCl. It contained MK-6 as the predominant menaquinone and iso-C(15 : 0), iso-C(15 : 1), iso-C(17 : 1) 3-OH, iso-C(15 : 0) 3-OH and iso-C(15 : 0) 2-OH as the major fatty acids. The genomic DNA G+C content was 31.3 mol%. On the basis of the phenotypic characteristics and phylogenetic distance, it can be concluded that strain KMM 9545(T) represents a novel species of the genus Tamlana, for which the name Tamlana sedimentorum sp. nov. is proposed. The type strain is KMM 9545(T) ( = NRIC 0921(T) = JCM 19808(T)).

Tolerance of Marine Proteobacteria of the Genera Pseudoalteromonasand Alteromonasto Heavy Metals

Most of metals [Li(I), Be(II), Al(III), V(IV), Cr(III), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II), Sr(II), Mo(VI), Ag(I), Cd(II), Sn(IV), Sb(III), Cs(I), Hg(II), Pb(II), U(VI), Pu(III)], which are abundant in the hydrosphere and atmosphere, occur in low concentrations (< 1 mmol/m 3 ) in freshwater and seawater [1]. The release of metals into the environment through the waste products generated by the metal-working industries leads to their accumulation in the hydrosphere and atmosphere and may affect the human health. Aquatic microbial communities, which are capable of self-regulation and adaptation to altered environments, may serve as suitable objects to assess the contamination level at which microorganisms still maintain their homeostasis [2, 3].

Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T

Fucoidans belong to a structurally heterogeneous class of sulfated polysaccharides isolated from brown algae. They have a wide spectrum of biological activities. The complex structures of these polysaccharides hinder structure-activity relationships determination. Fucoidan sulfatases can make useful tools for the determination of the fine chemical structure of fucoidans. In this study, identification and preparation of two recombinant sulfatases able to catalyze the cleavage of sulfate groups from fragments of fucoidan molecules is described for the first time. Two genes of sulfatases swf1 and swf4 of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T were cloned and the proteins were produced in Escherichia coli cells. Sulfatases SWF1 and SWF4 are assigned to S1_17 and S1_25 subfamilies of formylglycine-dependent enzymes of S1 family (SulfAtlas). Some molecular and biochemical characteristics of recombinant fucoidan sulfatases have been studied. Detailed specificity and catalytic features of sulfatases were determined using various sulfated fucooligosaccharides. Structures of products produced by SWF1 and SWF4 were established by nuclear magnetic resonance (NMR) spectroscopy. Based on the obtained data, the enzymes are classified as fucoidan exo-2O-sulfatase (SWF1) and fucoidan exo-3O-sulfatase (SWF4). In addition, we demonstrated the sequential action of sulfatases on 2,3-di-O-sulfated fucooligosacchrides, which indicates an exolitic degradation pathway of fucoidan by a marine bacterium W. fucanilytica CZ1127T.