Morphology, physiological characteristics, and complete sequence of marine bacteriophage ϕRIO-1 infecting Pseudoalteromonas marina.

Bacteria of the genus Pseudoalteromonas are ubiquitous in the world's oceans. Marine bacteria have been posited to be associated with a major ancient branch of podoviruses related to T7. Yet, although Pseudoalteromonas phages belonging to the Corticoviridae and the Siphoviridae and prophages belonging to the Myoviridae have been reported, no Pseudoalteromonas podovirus was previously known. Here, a new lytic Pseudoalteromonas marina phage, ϕRIO-1, belonging to the Podoviridae was isolated and characterized with respect to morphology, genomic sequence, and biological properties. Its major encoded proteins were distantly similar to those of T7. The most similar previously sequenced viruses were Pseudomonas phage PA11 and Salinivibrio phage CW02. Whereas many elements of the morphology and gene organization of ϕRIO-1 are similar to those of podoviruses broadly related to T7, ϕRIO-1 conspicuously lacked an RNA polymerase gene. Since definitions of a T7 supergroup have included similarity in the DNA polymerase gene, a detailed phylogenetic analysis was conducted, and two major DNA polymerase clades in Autographivirinae and several structural variants of the polA family represented in podoviruses were found. ϕRIO-1 carries an operon similar to that in a few other podoviruses predicted to specify activities related to γ-glutamyl amide linkages and/or unusual peptide bonds. Most growth properties of ϕRIO-1 were typical of T7-like phages, except for a long latent period.

The potential effect of botulinum toxin type A on human dermal fibroblasts: an in vitro study.

BACKGROUND: The use of botulinum toxin type A (BoNT) continues to expand. Some physicians have noted a face-lifting effect after intradermal injection of BoNT, although the effects are controversial. OBJECTIVE: To investigate the in vitro effects of BoNT on human dermal fibroblasts. METHODS: The proliferation and toxic effects of BoNT on human dermal fibroblasts were measured. To understand the mechanism of BoNT on collagen production of fibroblasts, procollagen type I carboxy-terminal peptide (PIP) was measured using enzyme-linked immunosorbent assay, and collagen production was monitored using Western blotting. To examine the effect of BoNT on collagen degradation, we evaluated matrix metalloproteinase (MMP) production using gelatin zymography. RESULTS: BoNT did not stimulate the proliferation of or show toxic effects on human dermal fibroblasts. Levels of PIP increased significantly in fibroblasts grown in the presence of BoNT, and BoNT upregulated the expression of type I collagen and decreased the production of some MMPs in fibroblasts that prevent collagen degradation. CONCLUSIONS: This study shows interesting effects of BoNT on collagen production and degradation of human dermal fibroblasts in vitro. This research provides the experimental background for using intradermal BoNT injection for remodeling of dermal tissues in aged skin.

Complete Genome Sequence of Sulfitobacter Phage ϕGT1, Isolated from a Tidal Flat.

The Sulfitobacter bacteria are ubiquitous and important players in organic sulfur cycling in marine environments. Here, we report the complete genome sequence of ϕGT1 infecting Sulfitobacter sp. HGT1, both of which were isolated from coastal sediment. ϕGT1 has a 40,019-bp genome containing 69 predicted protein-encoding genes.

Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938).

Although Pleurostomum was described almost a century ago, flagellates assigned to this taxon have been recorded only in very occasional faunistic studies of highly saline habitats, and their phylogenetic position has remained uncertain. We report the cultivation, ultrastructure, and phylogenetic relationships of Pleurostomum flabellatum isolated from a Korean saltern pond of 313 per thousand salinity. This isolate is biflagellated with a cytostomal groove, and is not distinguishable from previous accounts of P. flabellatum from saturated brines in India and Australia. Pleurostomum flabellatum shows ultrastructural features characteristic of many Heterolobosea: (1) a striated rhizoplast, (2) an absence of stacked Golgi bodies, (3) parallel basal bodies and flagella, and (4) a large number of peripheral microtubules supporting a rostrum. 18S rRNA gene phylogenies strongly confirm the affinities of P. flabellatum within Heterolobosea. Furthermore, the 18S rRNA gene of P. flabellatum has the heterolobosean-specific helix 17_1, and a group I intron in the same position as in Acrasis rosea. Within Heterolobosea, the 'amoeboflagellate' genera Naegleria and Willaertia were its closest relatives with high bootstrap support and posterior probability. P. flabellatum was observed only as a flagellate, and never as an amoeba. Since light microscopy and electron microscopy observations indicate that P. flabellatum flagellates are capable both of feeding and division, there might be no amoeba stage. Being morphologically distinct from its closest relatives and phylogenetically distant from other flagellate-only Heterolobosea, P. flabellatum cannot be moved into any previously described heterolobosean genus. Instead, we move Pleurostomum into Heterolobosea, and assign as the type species Pleurostomum salinum Namyslowski 1913, a species that closely resembles P. flabellatum. The optimal temperature for growth of P. flabellatum is 40 degrees C. Interestingly, P. flabellatum grows optimally at 300 per thousand salinity and fails to grow below 200 per thousand salinity, indicating that it is an 'extreme halophile'. The optimal salinity for growth is the highest for any eukaryote examined to date.

Identification of structural and morphogenesis genes of Pseudoalteromonas phage φRIO-1 and placement within the evolutionary history of Podoviridae.

The virion proteins of Pseudoalteromonas phage φRIO-1 were identified and quantitated by mass spectrometry and gel densitometry. Bioinformatic methods customized to deal with extreme divergence defined a φRIO-1 tail structure homology group of phages, which was further related to T7 tail and internal virion proteins (IVPs). Similarly, homologs of tubular tail components and internal virion proteins were identified in essentially all completely sequenced podoviruses other than those in the subfamily Picovirinae. The podoviruses were subdivided into several tail structure homology groups, in addition to the RIO-1 and T7 groups. Molecular phylogeny indicated that these groups all arose about the same ancient time as the φRIO-1/T7 split. Hence, the T7-like infection mechanism involving the IVPs was an ancestral property of most podoviruses. The IVPs were found to variably host both tail lysozyme domains and domains destined for the cytoplasm, including the N4 virion RNA polymerase embedded within an IVP-D homolog.

Marivita cryptomonadis gen. nov., sp. nov. and Marivita litorea sp. nov., of the family Rhodobacteraceae, isolated from marine habitats.

Two strictly aerobic, Gram-negative, rod-shaped bacteria containing photosynthesis-related genes, designated strains CL-SK44(T) and CL-JM1(T), were isolated from a culture of the marine phytoplankton Cryptomonas sp. and coastal seawater from Korea, respectively. Phylogenetic analysis of 16S rRNA gene sequences revealed that the two strains were related to members of the genera Thalassobius (95.3-96.7 % similarity), Pelagibaca (95.3-96.0 %) and Donghicola (95.6 %) in the family Rhodobacteraceae. However, the two novel strains did not form a robust clade with any species of the Roseobacter clade, forming a distinct clade. The major polar lipids of the strains were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unidentified aminolipid and an unidentified lipid, profiles that were distinguishable from those of the related genera examined. Although the level of 16S rRNA gene sequence similarity between strains CL-SK44(T) and CL-JM1(T) was very high (99.1 %), DNA-DNA relatedness between the strains was 13 %, suggesting that they represent genomically distinct species. In addition, the two strains could be differentiated based on the presence of a minor polar lipid, on the hydrolysis of gelatin and the utilization of carbon sources. Based on the data from the present study, strains CL-SK44(T) and CL-JM1(T) are considered to represent separate novel species of a new genus of the family Rhodobacteraceae, for which the names Marivita cryptomonadis gen. nov., sp. nov. (type species) and Marivita litorea sp. nov. are proposed. The type strains of Marivita cryptomonadis and Marivita litorea are CL-SK44(T) (=KCCM 90070(T)=JCM 15447(T)) and CL-JM1(T) (=KCCM 90071(T)=JCM 15446(T)), respectively.

Maritalea myrionectae gen. nov., sp. nov., isolated from a culture of the marine ciliate Myrionecta rubra.

A Gram-negative, rod-shaped, strictly aerobic bacterium, designated strain CL-SK30(T), was isolated from a culture of the marine ciliate Myrionecta rubra. 16S rRNA gene sequence analysis revealed that strain CL-SK30(T) was most closely related to Cucumibacter marinus (92.0 % similarity) and next to the type strains of species of the genus Devosia (89.8-91.3 % similarities) in the family Hyphomicrobiaceae. Phylogenetic analyses of the 16S rRNA gene sequences showed that strain CL-SK30(T) formed a robust clade together with C. marinus, but the sequence divergence value of 8 % between them indicated that the novel bacterium represented a distinct lineage. Strain CL-SK30(T) grew optimally in the presence of 2-5 % sea salts at 30-35 degrees C and pH 7.2-8.0. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, two unidentified glycolipids, an unidentified phospholipid and an unidentified lipid. Ubiquinone 10 was the major quinone. The DNA G+C content was 52.7 mol%. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain CL-SK30(T) represents a novel genus and species of the family Hyphomicrobiaceae, for which the name Maritalea myrionectae gen. nov., sp. nov. is proposed. The type strain is CL-SK30(T) (=KCCM 90060(T)=DSM 19524(T)).

Muricauda olearia sp. nov., isolated from crude-oil-contaminated seawater, and emended description of the genus Muricauda.

A Gram-staining-negative, rod-shaped and non-motile bacterium, designated CL-SS4T, was isolated from crude-oil-contaminated seawater from the west coast of Korea and was investigated by means of a polyphasic taxonomic approach. Strain CL-SS4T grew optimally at 25-30 degrees C, at pH 6.8-7.7 and in the presence of 2-3% sea salts. The major fatty acids were iso-C17:0 3-OH, iso-C15:0 and iso-C15:1 G. The major isoprenoid quinone was MK-6. The DNA G+C content was 50.7 mol%. Analysis of the 16S rRNA gene sequence of strain CL-SS4T revealed that it was a member of the genus Muricauda, sharing 95.7-96.9% sequence similarity with the type strains of Muricauda species and being most closely related to Muricauda aquimarina SW-63T. Phylogenetic analyses based on 16S rRNA gene sequences confirmed that strain CL-SS4T formed a deep lineage related to the genus Muricauda. Strain CL-SS4T was distinguishable from recognized Muricauda species on the basis of differential phenotypic characteristics and DNA G+C content. Based on these results, it is proposed that strain CL-SS4T represents a novel species of the genus Muricauda, for which the name Muricauda olearia sp. nov. is proposed. An emended description of the genus Muricauda is also proposed. The type strain is CL-SS4T (=KCCM 90075T=JCM 15563T).

Gracilimonas tropica gen. nov., sp. nov., isolated from a Synechococcus culture.

An irregular, long, rod-shaped marine bacterium, designated CL-CB462(T), was isolated from a Synechococcus culture, which was established from surface water from the tropical Pacific Ocean. The physiological and biochemical features, fatty acid profile and phylogenetic position based on 16S rRNA gene sequences were investigated for the novel strain. Phylogenetic analysis of the 16S rRNA gene sequence showed that the closest relatives of strain CL-CB462(T) were Balneola vulgaris and Balneola alkaliphila. Strain CL-CB462(T) formed a robust clade with members of the genus Balneola in all phylogenetic trees constructed by three different methods. However, the 16S rRNA gene sequence similarity was very low (91.3-91.5 % similarity) and phenotypic and physiological features could clearly differentiate strain CL-CB462(T) from the genus Balneola. Cells of the novel strain were non-motile and spore-forming. The strain was able to grow at 1-20 % (w/v) (optimum of 3-6 %) sea salt concentration, at temperatures of 20-40 degrees C and between pH 6 and 10. The fatty acids were dominated by 15 : 0 iso (41.2 %) and 17 : 1omega9c iso (21.4 %). The DNA G+C content was 42.7 mol%. Based on polyphasic evidence, strain CL-CB462(T) was considered to represent a new genus. The name Gracilimonas tropica gen. nov., sp. nov. is proposed for the type strain of the type species (CL-CB462(T)=KCCM 90063(T)=DSM 19535(T)).

Cucumibacter marinus gen. nov., sp. nov., a marine bacterium in the family Hyphomicrobiaceae.

A Gram-negative, rod-shaped, strictly aerobic micro-organism, designated strain CL-GR60(T), was isolated from coastal seawater from the East Sea, Korea. A 16S rRNA gene sequence analysis revealed a clear affiliation with the family Hyphomicrobiaceae. Phylogenetic analyses showed that strain CL-GR60(T) formed a robust cluster with the species of the genus Devosia at sequence similarity levels of 91.1-93.1 %; no other species in the family Hyphomicrobiaceae shared more than 90 % sequence similarity with strain CL-GR60(T). The strain grew optimally in the presence of 3-4 % sea salts at 30-35 degrees C and pH 7. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, two unidentified glycolipids, an unidentified phospholipid and an unidentified lipid. The strain contained C(18 : 1)omega7c (54.7 %), 11-methyl C(18 : 1)omega7c (19.3 %), C(18 : 0) (14.4 %) as the predominant fatty acids. Ubiquinone-10 was the major quinone. The DNA G+C content was 62.9 mol%. On the basis of physiological data, fatty acid composition and results of phylogenetic analysis of the 16S rRNA gene sequence, strain CL-GR60(T) represents a novel genus and species within the family Hyphomicrobiaceae, for which the name Cucumibacter marinus gen. nov., sp. nov. is proposed. The type strain of Cucumibacter marinus is CL-GR60(T) (=KCCM 90027(T) =DSM 18995(T)).

Thalassobaculum litoreum gen. nov., sp. nov., a member of the family Rhodospirillaceae isolated from coastal seawater.

A Gram-negative, facultatively anaerobic strain with slightly curved and straight rod-shaped cells, strain CL-GR58(T), was isolated from coastal seawater (near Gori, Korea). Analyses of the 16S rRNA gene sequence revealed that strain CL-GR58(T) belonged to the family Rhodospirillaceae with Azospirillum lipoferum as its closest relative (gene sequence similarity of 90.9 %). Phylogenetic analyses of the 16S rRNA gene sequences showed that strain CL-GR58(T) was not associated with any known genera in the family Rhodospirillaceae. The novel strain grew in the presence of 1-10 % sea salts, optimally at 30-35 degrees C and pH 8. The major cellular fatty acids consisted of C(18 : 1)omega7c (48.5 %), C(16 : 0) (14.8 %), C(17 : 0) (12.2 %), C(19 : 0) cyclo omega8c (6.3 %) and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH, 6.0 %). Among the phylogenetically related genera, the fatty acid C(17 : 0) was found only in strain CL-GR58(T). The DNA G+C content of the novel strain was 68.0 mol%. According to phylogenetic analyses of the 16S rRNA gene sequence, fatty acid content and the physiological data, strain CL-GR58(T) represents a novel species in a new genus of the family Rhodospirillaceae, for which the name Thalassobaculum litoreum gen. nov., sp. nov. is proposed. The type strain of the type species is CL-GR58(T) (=KCCM 42674(T)=DSM 18839(T)).

Maribius salinus gen. nov., sp. nov., isolated from a solar saltern and Maribius pelagius sp. nov., cultured from the Sargasso Sea, belonging to the Roseobacter clade.

Two strictly aerobic, Gram-negative bacteria, designated strains CL-SP27T and B5-6T, were isolated from the hypersaline water of a solar saltern in Korea and from the surface water of the Sargasso Sea, respectively. The two strains were rod-shaped, non-motile and grew on marine agar 2216 as beige colonies. Phylogenetic analyses of 16S rRNA gene sequences revealed a clear affiliation of the novel strains to the family Rhodobacteraceae. However, the novel strains were only distantly related to members of the Roseobacter clade, forming a distinct lineage. Although the 16S rRNA gene sequence similarity between strains CL-SP27T and B5-6T was very high (99.6 %), DNA-DNA relatedness between the strains was 48.4 %, suggesting that the strains be categorized as two genospecies. Additionally, the two novel strains could be differentiated by DNA G+C contents, fatty acid profiles, carbon source utilization patterns, antibiotic susceptibilities and biochemical characteristics. Based on taxonomic data obtained in this study, strains CL-SP27T and B5-6T represent separate species within a novel genus of the family Rhodobacteraceae, for which the names Maribius salinus gen. nov., sp. nov. (type species) and Maribius pelagius sp. nov. are proposed. The type strains of Maribius salinus and Maribius pelagius are CL-SP27T (=KCCM 42113T=JCM 13037T) and B5-6T (=KCCM 42336T=JCM 14009T), respectively.

Citreimonas salinaria gen. nov., sp. nov., a member of the Roseobacter clade isolated from a solar saltern.

A lemon-shaped marine bacterium, strain CL-SP20(T), isolated from hypersaline water from a solar saltern in Korea, was characterized in terms of its physiological and biochemical features, its fatty acid profile and its phylogenetic position based on 16S rRNA gene sequences. Analysis of the 16S rRNA gene sequence revealed a clear affiliation with the Roseobacter lineage (91.0-96.3 % similarity) of the family Rhodobacteraceae. However, strain CL-SP20(T) did not form a robust clade with any species of the Roseobacter clade, forming a distinct subline. Strain CL-SP20(T) is non-motile and forms beige colonies on marine agar. The strain is able to grow with sea salts at concentrations in the range 1-10 %, with optimal growth between 5 and 6 %. It grows at temperatures in the range 15-40 degrees C and at pH 6-10. The strain cannot oxidize thiosulfate. The fatty acids are dominated by 18 : 1omega7c (54.3 %) and 19 : 0 cyclo omega8c (20.4 %). The DNA G+C content is 67.3 mol%. According to the physiological data, fatty acid composition and phylogenetic analysis of the 16S rRNA gene sequence, strain CL-SP20(T) represents a novel species in a novel genus of the family Rhodobacteraceae, for which the name Citreimonas salinaria gen. nov., sp. nov. is proposed. The type strain of Citreimonas salinaria is CL-SP20(T) (=KCCM 42116(T)=JCM 13036(T)).

Lutibacter litoralis gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment.

A rod-shaped marine bacterium, designated strain CL-TF09T, isolated from a tidal flat in Ganghwa, Korea, was characterized based on its physiological and biochemical features, fatty acid profile and phylogenetic position. 16S rRNA gene sequence analysis revealed a clear affiliation with the family Flavobacteriaceae. Strain CL-TF09T showed the closest phylogenetic relationship with the genera Tenacibaculum and Polaribacter; sequence similarities between CL-TF09T and the type strains of Tenacibaculum and Polaribacter species ranged from 90.7 to 91.8 %. Cells of strain CL-TF09T were non-motile and grew on solid media as yellow colonies. The strain grew in the presence of 1-5 % sea salts, within a temperature range of 5-30 degrees C and at pH 7-8. The strain had iso-C(15 : 0) 3-OH (17.4 %), iso-C(15 : 0) (16.7 %), anteiso-C(15 : 0) (15.1 %) and iso-C(16 : 0) 3-OH (13.4 %) as predominant fatty acids. The DNA G+C content was 33.9 mol%. Based on the physiological, fatty acid composition and phylogenetic data presented, strain CL-TF09T is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Lutibacter litoralis gen. nov., sp. nov. is proposed. The type strain is CL-TF09T (=KCCM 42118T = JCM 13034T).

Flectobacillus lacus sp. nov., isolated from a highly eutrophic pond in Korea.

A Gram-negative, almost straight, rod-shaped, non-motile bacterium, designated CL-GP79(T), was isolated from a highly eutrophic pond located within the campus of Seoul National University, Korea. Phylogenetic analyses based on 16S rRNA gene sequencing showed that strain CL-GP79(T) belongs to the family Flexibacteraceae, with Flectobacillus major as its closest relative (similarity of 95.7 %). The cellular fatty acids consist mainly of C(16 : 1)omega5c (mean +/- sd, 26.9 +/- 10.8 %), iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (19.2 +/- 2.3 %) and iso-C(15 : 0) (12.1 +/- 1.3 %). The DNA G + C content was found to be 38.3 mol%. Phenotypic, chemotaxonomic and phylogenetic analyses indicated that strain CL-GP79(T) could be assigned to the genus Flectobacillus, but could be distinguished from F. major. The strain CL-GP79(T) therefore represents a novel species, for which the name Flectobacillus lacus sp. nov. is proposed, with CL-GP79(T) (=KCCM 42271(T)=JCM 13398(T)) as the type strain.

Shimia marina gen. nov., sp. nov., a novel bacterium of the Roseobacter clade isolated from biofilm in a coastal fish farm.

A rod-shaped marine bacterium, CL-TA03(T), isolated from a biofilm in a coastal fish farm in Tongyeong, Korea, was characterized for physiological and biochemical features, fatty acid profile and phylogenetic position based on 16S rRNA gene sequences. Analysis of the 16S rRNA gene sequence revealed a clear affiliation with the family Rhodobacteraceae. Phylogenetic analysis of the 16S rRNA gene sequence showed that the closest relatives of CL-TA03(T) were Thalassobius gelatinovorus and Thalassobius mediterraneus (95.6 % similarity). The sequence similarities between CL-TA03(T) and other type species of the Roseobacter lineage ranged from 92.4 to 95.4 %. Strain CL-TA03(T) is motile and grows on marine agar as colourless or beige colonies. The strain is able to grow optimally in the range of 3-5 % sea salts. It grows within a temperature range of 15-35 degrees C and at pH 6-10. The fatty acids are dominated by 18 : 1omega7c (64.1 %) and 11-methyl 18 : 1omega7c (10.6 %). The DNA G+C content is 57.2 mol%. According to physiological data, fatty acid composition and phylogenetic analysis of the 16S rRNA gene sequence, CL-TA03(T) is considered to represent a new genus in the family Rhodobacteraceae and the name Shimia marina gen. nov., sp. nov. is proposed. The type strain of Shimia marina is CL-TA03(T) (=KCCM 42117(T)=JCM 13038(T)).

Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern.

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300 per thousand salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30-35 degrees C. The isolated HNF grew optimally at 150 per thousand salinity and tolerated up to 363 per thousand salinity, but it failed to grow below 75 per thousand salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.