Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium, sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.

5,7-Dihydroxy-5,6,7,8-tetrahydro-1H-azocin-2-one from a marine-derived Streptomyces sp.

In the course of a screening program, we have isolated the new natural product, 5,7-dihydroxy-5,6,7,8-tetrahydroazocin-2(IH)-one (1), from the staurosporine producing marine-derived Streptomyces sp. strain QD518. Here we report the isolation and structure elucidation of 1 and the artifacts 3 and 4 resulting from I by acid catalyzed intra- and inter-molecular reactions.

Heterologous expression and purification of recombinant allophycocyanin in marine Streptomyces sp isolate M097

Allophycocyanin is one of the most important marine active peptides. Previous studies suggested that recombinant allophycocyanin (rAPC) could remarkably inhibit the S-180 carcinoma in mice, indicating its potential pharmaceutical uses. Based on intergeneric conjugal transfer, heterologous expression of rAPC was first achieved in marine Streptomyces sp. isolate M097 through inserting the apc gene into the thiostrepton-induced vector pIJ8600. The transformation frequency for this system was approximately 10(-4) exconjugants/recipient. In the transformed Streptomyces sp. isolate M097, the yield of purified rAPC could amount to about 38 mg/l using a simple purification protocol, and HPLC analysis showed that the purity of the protein reached about 91.5%. In vitro activity tests also revealed that the purified rAPC had effective scavenging abilities on superoxide and hydroxyl radicals. This would widen the usefulness of the marine Streptomyces as a host to express the rAPC and to offer industrial strain for the production of rAPC.

Purification and characterization of two types of chitosanase from a Microbacterium sp.

Two extracellular chitosanases (ChiX and ChiN) were extracted from Microbacterium sp. OU01 with Mr values of 81 kDa (ChiX) and 30 kDa (ChiN). ChiN was optimally active at pH 6.2 and 50 degrees C and ChiX at pH 6.6 and 60 degrees C (assayed over 15 min). Both the activities increased with the degree of deacetylation (DDA) of chitosan. ChiN hydrolyzed oligomers of glucosamine (GlcN) larger than chitopentaose, and chitosan with 62-100% DDA; but ChiX acted on chitosan and released GlcN. Hydrolysis of chitosan with 99% DDA by ChiN released chitobiose, chitotriose and chitotetraose as the major products.

Novel antifouling and antimicrobial compound from a marine-derived fungus Ampelomyces sp.

In this study, using a bioassay-guided isolation and purification procedure, we obtained 3-chloro-2,5-dihydroxybenzyl alcohol from a marine-derived Ampelomyces species that effectively inhibited larval settlement of the tubeworm Hydroides elegans and of cyprids of the barnacle Balanus amphitrite. The inhibitive effect on larval settlement was nontoxic and the EC50 of 3-chloro-2,5-dihydroxybenzyl alcohol ranged from 3.19 mu g ml(-1) to 3.81 mu g ml(-1) while the LC50 was 266.68 lambda g ml(-1) for B. amphitrite cyprids; EC50 ranged from 0.67 mu g ml(-1) to 0.78 mu g ml(-1), and LC50 was 2.64 mu g ml(-1) for competent larvae of H. elegans, indicating that inhibitive effect of this compound was nontoxic. At a concentration of 50 mu g per disc, this compound showed strong inhibitive effects on the growth of 13 out of 15 marine bacterial species tested in disc diffusion bioassay. Overall, the high inhibitory activities against bacteria and larval settlement as well as the non- or low-toxic nature of this compound to the barnacle and polychaete larvae suggest this compound could be a potent antifoulant and/or antibiotic.

attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.

Wangia profunda gen. nov., sp nov., a novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa Trough deep-sea sediment

An orange-pigmented, Gram-negative, nonmotile, strictly aerobic and oxidase- and catalase-positive bacterium (SM-A87(T)) was isolated from the deep-sea sediment of the southern Okinawa Trough area. The main fatty acids were i15 : 0, i17 : 0 3OH, i15 : 1 G, i17 : 1 omega 9c, 15 : 0, i15 : 0 3OH and summed feature 3 (comprising i-15 : 0 2OH and/or 16 : 1 omega 7c). MK-6 was the predominant respiratory quinone. DNA G+C content was 35.8 mol%. Flexirubin-type pigments were absent. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain SM-A87(T) formed a distinct lineage within the family Flavobacteriaceae, with < 93% sequence similarity to the nearest strain of genus Salegentibacter. Moreover, strain SM-A87(T) could be distinguished from the nearest phylogenetic neighbors by a number of chemotaxonomic and phenotypic properties. On the basis of polyphasic analyses, it is proposed that strain SM-A87(T) be classified in a novel genus and a new species in the family Flavobacteriaceae, designated Wangia profunda gen. nov., sp. nov. The type strain is SM-A87(T) (CCTCC AB 206139(T)=DSM 18752).

Myroides profundi sp nov., isolated from deep-sea sediment of the southern Okinawa Trough

A Gram-negative, nonmotile, aerobic and oxidase- and catalase-positive bacterium,, designated D25(T), was isolated from the deep-sea sediments of the southern Okinawa Trough area. Phylogenetic analyses of 16S rRNA gene sequences showed that strain D25(T), fell within the genus Myroides, with 99.2%, 96.0% and 93.4% sequence similarities to the only three recognized species of Myroides. However, the DNA-DNA similarity Value between strain D25(T) and its nearest neighbour Myroides odoratimimus JCM 7460(T) was only 49.9% ( < 70%). Several phenotypic properties could be used to distinguish strain D25(T) from other Myroides species. The main cellular fatty acids of strain D25(T) were iso-C-15:0, iso-C-17:1 omega 9C, iso-C(17:0)3-OH and Summed Feature 3 (comprising C-16:1 omega 7c and/or iso-C(15:0)2-OH). The major respiratory quinone was MK-6. The DNA G+C content was 33.0 mol%. The results of the polyphasic taxonomy analysis suggested that strain D251(T) represents a novel species of the genus Myroides, for which the name Myroides profundi sp. nov. is proposed. The type strain is D25(T) (=CCTCC M 208030(T) = DSM 19823(T)).

Two different proteases produced by a deep-sea psychrotrophic bacterial strain, Pseudoaltermonas sp SM9913

A psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913, was isolated from deep-sea sediment collected at 1,855 m depth. Two proteases produced by Pseudoaltermonas sp. SM9913 were purified, MPC-01 and MCP-02. MCP-01 is a serine protease with a molecular weight of 60.7 kDa. It is cold-adapted with an optimum temperature of 30-35degreesC. Its K-m and E-a for the hydrolysis of casein were 0.18% and 39.1 kJ mol(-1), respectively. It had low thermostability, and its activity was reduced by 73% after incubation at 40degreesC for 10 min. MCP-02 is a mesophilic metalloprotease with a molecular weight of 36 kDa. Its optimum temperature for the hydrolysis of casein was 50-55degreesC. The K-m and E-a of MCP-02 for the hydrolysis of casein were 0.36% and 59.3 kJ mol(-1), respectively. MCP-02 had high thermostability, and its activity was reduced by only 30.5% after incubation at 60degreesC for 10 min. At low temperatures, Pseudoaltermonas sp. SM9913 mainly produced the psychrophilic protease MCP-01.

Holosticha hamulata n. sp and Holosticha heterofoissneri Hu and Song, 2001, two urostylid ciliates (Protozoa, Ciliophora) from intertidal sediments of the Yellow Sea

Two marine urostylid ciliates, Holosticha hamulata n. sp. and Holosticha heterofoissneri Hu and Song, 2001, were investigated using live observation and protargol impregnation. Both species were isolated from Korean intertidal sediments of the Yellow Sea. Holosticha hamulata measures about 150 x 25 pro in vivo, and is characterized by a tripartite body shape with a narrow head, an inflated trunk, and a tail that distally projects ventrally forming a hook-like structure. It is the characteristic body shape that distinguishes H. hamulata distinctly from congeners. Holosticha hamulata differs from H. heterofoissneri, possibly the nearest relative, also by the location of the contractile vacuole (ahead of mid-body versus near posterior body third) and the configuration of the macronucleus (on average, 33 scattered nodules assuming a Y-shape versus 17 nodules that may form a U shape). The average number of the macronuclear nodules is a pronounced feature showing great consistency in populations of each species. However, their arrangement is variable in H. heterofoissneri where the nodules are basically scattered or connected by fine fibers forming an elongate U-shape. The location of the contractile vacuole as a taxonomic feature is discussed and a dichotomous key to the species of Holosticha sensu stricto is provided.

Morphology and infraciliature of three species of Metaurostylopsis (Ciliophora, Stichotrichia): M. songi n. sp., M. salina n. sp., and M. marina (Kahl 1932) from sediments, saline ponds, and coastal waters.

Two new urostylid ciliates, Metaurostylopsis songi n. sp. and Metaurostylopsis salina n. sp. and Metaurostylopsis marina (Kahl 1932) are investigated using live observation and protargol impregnation. These species were isolated in Korea from intertidal sediments, saline ponds, and coastal waters. Metaurostylopsis songi is in vivo about 120 pm x 25 mu m, has a slenderly ellipsoidal body, colorless cortical granules in rows on ventral and dorsal body sides, about 54 macronuclear nodules, 28-47 adoral membranelles, five frontal, two or three frontoterminal and six or seven transverse cirri, and 9-12 midventral cirral pairs followed posteriorly by 1-3 single cirri. In vivo M. salina is about 60 pin x 25 mu m, has a pyriform body, colorless cortical granules irregularly arranged, about 45 macronuclear nodules, 18-23 adoral membranelles, three frontal, three to five frontoterminal and two to five transverse cirri, and four or five midventral cirral pairs followed posteriorly by five to seven single cirri. Both species have three marginal cirral rows on each body side and 3 long dorsal kineties. The Korean specimens of M. marina match the Chinese population in all main features. Metaurostylopsis songi differs from M. marina by the more slender body, the number of frontal cirri (invariably five vs. four), and the arrangement of cortical granules (in rows on dorsal and ventral cortex vs. only along dorsal kinetics and anterior body margin). Metaurostylopsis salina differs from its congeners by the distinctly smaller size, the pyriform body shape, the scattered cortical granules (vs. in rows), and number of frontal cirri. It differs from M. marina also by the number of midventral cirral pairs (four or five vs. seven to 11).

Descriptions of Protospathidium serpens (Kahl, 1930) and P-fraterculum n. sp (Ciliophora, Haptoria), two species based on different resting cyst morphology

Protospathidium serpens (Kahl, 1930) is frequent in semiterrestrial and terrestrial habitats worldwide. Conventionally, all populations are considered as conspecific because they have very similar overall morphologies and morphometrics. We studied in detail not only the morphology of the vegetative cells but also the resting cysts using live observation, protargol impregnation, and scanning electron microscopy. These revealed a cryptic diversity and biogeographic pattern in details of the dorsal brush and cyst wall morphology. The cyst wall is spiny in the Austrian specimens, while smooth in the South African and Antarctic populations. Accordingly, P. serpens consists of at least two species: P. serpens (with spiny cyst wall) and P. fraterculum n. sp. (with smooth cyst wall); the latter is probably composed of two distinct taxa differing by the absence (South African)/presence (Antarctic) of a monokinetidal bristle tail in brush row 3, the number of dikinetids comprising brush row 1 (seven versus three), and the total number of brush dikinetids (29 versus 17). Protospathidium serpens is neotypitied with the new population from Austria. The significance of resting cyst morphology is discussed with respect to alpha-taxonomy and overall ciliate diversity.

Laurencia nanhaiense sp nov., a new species of the genus Laurencia(Rhodomelaceae, Rhodophyta) from China

Laurencia nanhaiense sp. nov. (Rhodomelaceae, Rhodophyta) is described from Hainan and Guangdong Provinces, China. The new species clearly displays one of the defining features of the genus, viz. four periaxial cells per vegetative axial segment. It differs from other closely related species in having a combination of features such as terete axes from a basal system composed of a primary, discoid holdfast and a secondary attachment to give rise to many short rhizoids, branching oppositely and alternately, irregularly tristichous or subverticillately polystichous, having more curve branches with very sparse, adventitious ultimate branchlets, non-projecting superficial cortical cells at the apices of ultimate branchlets, presence of longitudinally oriented secondary pit-connections between contiguous superficial cortical cells, absence of lenticular thickenings in the walls of medullary cells, parallel arrangement of tetrasporangia along the axis of stichidia, and presence of intercellular spaces between medullary cells.

Two trichodinid ectoparasites from marine molluscs in the Yellow Sea, off China, with the description of Trichodina caecellae n. sp (Protozoa : Ciliophora : Peritrichia)

The morphology and infraciliature of two ectoparasitic ciliates, Trichodina caecellae n. sp. and T. ruditapicis Xu, Song & Warren, 2000, parasitising the gills of marine molluscs from the Shandong coast of the Yellow Sea, China, were investigated following wet silver nitrate and protargol impregnation. T. caecellae was found on the small marine sand clam Caecella chinensis Deshayes and is distinguished mainly by the acute triangle-like blade, the very delicate central part and the needle-shaped ray. T. ruditapicis was studied based on four populations from three clams: two populations from Ruditapes philippinarum (Adams) and one each from Saxidomus purpuratus (Sowerby) and Solen grandis Dunker. All four populations fell within the range of morphometry and agreed closely in the overall appearance of the adhesive disc. However, variability was found in the denticle structure, especially in populations from different host clams. Our observations suggest that denticle morphology may be more or less variable between and within populations, and that such minor differences should not be overestimated. It should be emphasised that, except for the denticle morphology, the bright granules or circles in the centre of the adhesive disc represent another important feature facilitating the identification of this trichodinid species.

Morphology and Molecular Phylogeny of Trimyema koreanum n. sp., a Ciliate from the Hypersaline Water of a Solar Saltern

A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293 parts per thousand) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 x 13 mu m in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).