Living in a bottle: Bacteria from sediment-associated Mediterranean waste and potential growth on polyethylene terephthalate.

Ocean pollution is a worldwide environmental challenge that could be partially tackled through microbial applications. To shed light on the diversity and applications of the bacterial communities that inhabit the sediments trapped in artificial containers, we analyzed residues (polyethylene terephthalate [PET] bottles and aluminum cans) collected from the Mediterranean Sea by scanning electron microscopy and next generation sequencing. Moreover, we set a collection of culturable bacteria from the plastisphere that were screened for their ability to use PET as a carbon source. Our results reveal that Proteobacteria are the predominant phylum in all the samples and that Rhodobacteraceae, Woeseia, Actinomarinales, or Vibrio are also abundant in these residues. Moreover, we identified marine isolates with enhanced growth in the presence of PET: Aquimarina intermedia, Citricoccus spp., and Micrococcus spp. Our results suggest that the marine environment is a source of biotechnologically promising bacterial isolates that may use PET or PET additives as carbon sources.

X-ray Crystallographic Structure and Oligomerization of Gloeobacter Rhodopsin.

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR's hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR's oligomerization state using double electron-electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.

Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors.

Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.

The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation.

Conserved C-terminal domains (CTD) have been shown to act as a signal for the translocation of certain proteins across the outer membrane of Bacteroidetes via a type IX secretion system (T9SS). The genome sequence of the periodontal pathogen Tannerella forsythia predicts the presence of the components for a T9SS in conjunction with a suite of CTD proteins. T. forsythia is covered with a two-dimensional crystalline surface (S-) layer composed of the glycosylated CTD proteins TfsA and TfsB. To investigate, if T9SS is functional in T. forsythia, T9SS-deficient mutants were generated by targeting either TF0955 (putative C-terminal signal peptidase) or TF2327 (PorK ortholog), and the mutants were analyzed with respect to secretion, assembly and glycosylation of the S-layer proteins as well as proteolytic processing of the CTD and biofilm formation. In either mutant, TfsA and TfsB were incapable of translocation, as evidenced by the absence of the S-layer in transmission electron microscopy of ultrathin-sectioned bacterial cells. Despite being entrapped within the periplasm, mass spectrometry analysis revealed that the S-layer proteins were modified with the complete, mature glycan found on the secreted proteins, indicating that protein translocation and glycosylation are two independent processes. Further, the T9SS mutants showed a denser biofilm with fewer voids compared with the wild-type. This study demonstrates the functionality of T9SS and the requirement of CTD for the outer membrane passage of extracellular proteins in T. forsythia, exemplified by the two S-layer proteins. In addition, T9SS protein translocation is decoupled from O-glycan attachment in T. forsythia.

Paludibacter jiangxiensis sp. nov., a strictly anaerobic, propionate-producing bacterium isolated from rice paddy field.

A mesophilic, obligately anaerobic, propionate-producing fermentative bacterium, designated strain NM7(T), was isolated from rural rice paddy field. Cells of strain NM7(T) are Gram-negative, non-motile, non-spore-forming, short rods, and negative for catalase. The strain grew optimally at 37 °C (the range for growth 15-40 °C) and pH 7.0 (pH 5.0-7.5). The strain could grow fermentatively on various sugars, including arabinose, xylose, fructose, galactose, glucose, mannose, cellobiose, lactose, maltose, sucrose, pectin and starch. The main end products of glucose fermentation were acetate and propionate. Yeast extract was not required but stimulated the growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, and Fe(III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of genomic DNA was 42.8 mol%. The major cellular fatty acids were C15:0, anteiso-C15:0, C16:0, and C17:0. The most abundant polar lipid of strain NM7(T) was phosphatidylethanolamine. 16S rRNA gene sequence analysis revealed that it belongs to the family Porphyromonadaceae of the phylum Bacteroidetes. The closest recognized species was Paludibacter propionicigenes (91.4 % similarity in 16S rRNA gene sequence). A novel species, Paludibacter jiangxiensis sp. nov., is proposed to accommodate strain NM7(T) (=JCM 17480(T) = CGMCC 1.5150(T) = KCTC 5844(T)).

Marinifilum flexuosum sp. nov., a new Bacteroidetes isolated from coastal Mediterranean Sea water and emended description of the genus Marinifilum Na et al., 2009.

A facultatively anaerobe, moderately halophilic, Gram-negative, filamentous, non motile and unpigmented bacterium, designated M30(T), was isolated from coastal Mediterranean Sea water in Valencia, Spain. Phylogenetic analysis based on 16S rRNA sequences placed this strain in the phylum "Bacteroidetes" with Marinifilum fragile JC2469(T) as its closest relative with 97% sequence similarity. Average nucleotide identity (ANI) values between both strains were far below the 95% threshold value for species delineation (about 89% using BLAST and about 90% using MUMmer). A comprehensive polyphasic study, including morphological, biochemical, physiological, chemotaxonomic and phylogenetic data, confirmed the independent species status of strain M30(T) within the genus Marinifilum, for which the name Marinifilum flexuosum sp. nov. is proposed. The type strain of Marinifilum flexuosum is M30(T) (=CECT 7448(T)=DSM 21950(T)).

Analysis of the cell surface layer ultrastructure of the oral pathogen Tannerella forsythia.

The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium.

Rubidimonas crustatorum gen. nov., sp. nov., a novel member of the family Saprospiraceae isolated from a marine crustacean.

A strictly aerobic, Gram-negative, reddish-orange pigmented, non-motile and rod-shaped bacterium, designated AK17-053(T) was isolated from a marine crustacean (Squillidae) living on tidal flats on the coast of the Ariake Sea, Nagasaki, Japan. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate could be affiliated with the family Saprospiraceae of the phylum Bacteroidetes and that it showed highest sequence similarity (84%) with Lewinella marina MKG-38(T). The strain could be differentiated phenotypically from recognized members of the family Saprospiraceae. The G+C content of DNA was 55.3 mol%, MK-7 was the major menaquinone and iso-C(15:0) and C(16:1)ω7c were the major fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strain AK17-053(T) represents a new genus of the family Saprospiraceae. We propose the name Rubidimonas crustatorum gen. nov., sp. nov. for this strain; its type strain is AK17-053(T) (= MBIC08356(T) = NBRC 107717(T)).

A Cardinium-like symbiont in the proturan Acerella muscorum (Hexapoda).

Endosymbionts of the Cardinium-like genus are described in the testes and other tissues of the proturan Acerella muscorum (Ionescu). Few endosymbionts are present in the large apical cells of functional testes, but they become numerous at the end of the reproductive cycle. They are also found within sperm cells where induce their degeneration. The Gram-negative endosymbionts are characterized by the presence of microtubule-like structures (MLC) in their cytoplasm. It is suggested a possible role of the endosymbionts in the elimination of degenerating sperm cells when the testes activity is ended, thus somewhat playing a role in the timing of the reproductive cycle of the proturan species.

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana.

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the alpha-Proteobacteria (Ochrobactrum symbiont), beta-Proteobacteria (Beta-1 and Beta-2 symbionts), delta-Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense beta-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.

Multiple symbiosis in the leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae): details of transovarial transmission of Cardinium sp. and yeast-like endosymbionts.

Scaphoideus titanus is the insect vector of flavescence dorée (FD), a yellow disease of grapevines. Observations on adult females and nymphs of S. titanus showed that this insect is associated with a complex microbial community. Ultrastructural analysis showed that the fat body, salivary glands and ovary of the insect harbour microorganisms showing the brush-like structure typically observed in the genus Cardinium. In particular, it has been shown that these symbiotic bacteria are present both in the follicular cells and in the eggs. In addition, cells resembling bacteriocytes, harbouring numerous Cardinium symbionts in the cytoplasm, were observed in the apical portion of the ovary in adult females. These cells are likely responsible for bacterial transmission to the ovary. Optical microscopy showed that the fat body harbours an enormous population of yeast-like symbionts (YLSs). Ultrastructural observations showed that these symbionts are enclosed within specialized cells of the fat body and are also present in the ovary, where they are found in both the follicular cells and the eggs. There is thus evidence that both Cardinium and the YLSs are transovarially transmitted to the offspring. To our knowledge, S. titanus is the sole insect known to transmit two different kinds of symbionts to the eggs, a prokaryote and an eukaryote. Gene sequence analysis and in situ hybridization led to the identification of YLSs as members of the class Sordariomycetes (=Pyrenomycetes). Finally, ultrastructural observation of the midgut content revealed the presence, in both adult females and nymphs, of a complex microbial community, which include a phytoplasma-like microorganism, likely the agent of FD.

Uncultivated Tannerella BU045 and BU063 are slim segmented filamentous rods of high prevalence but low abundance in inflammatory disease-associated dental plaques.

Uncultivated clones BU045 and BU063 and Tannerella forsythia, a 'consensus periodontal pathogen', are the closest known relatives within the genus Tannerella. They have been described to inhabit different ecological niches of the human oral cavity. In this study, fluorescent in situ hybridization (FISH) and immunofluorescence were combined to investigate the prevalence and abundance of BU045 and BU063 in comparison to T. forsythia in plaques from gingivitis, necrotizing ulcerative gingivitis (NUG) and chronic periodontitis. Phylotype-specific FISH probes identified BU045 and BU063 as elongated thin rods with a segmented structure. Two structurally similar and previously unknown, rare phylotypes (127+ and 997+) were also identified due to partial 16S rRNA sequence identity with T. forsythia. In gingivitis, NUG and periodontitis patients, BU045, BU063, 127+, 997+ and T. forsythia were detected with prevalences of 50/83/71/14 and 81%, 100/100/86/17 and 53%, and 100/100/12/0 and 100%, respectively. Supragingivally, colonization density of all five organisms was generally low, rarely exceeding 0.1% of the total biota. In periodontal pocket samples, however, cell numbers of T. forsythia, but not of the uncultivable phylotypes, were greatly elevated. Our data demonstrate that Tannerella phylotypes BU045, BU063, 127+ and 997+ consist of long slim rods with segments, which, with respect to FISH stainability, often behaved as independent units. The phylotypes are frequent but low-level colonizers of various periodontal disease-associated plaques. Their apparent inability to proliferate to high density seems to exclude any relevance for the pathogenesis of periodontal diseases.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong.

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Loss of adherence ability to human gingival epithelial cells in S-layer protein-deficient mutants of Tannerella forsythensis.

Tannerella forsythensis, one of the important pathogens in periodontal disease, has a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. The S-layer in T. forsythensis is suggested to be associated with haemagglutinating activity, adhesion and invasion of host cells; however, its precise functions have been unknown. ORFs encoding the major S-layer proteins (230 and 270 kDa) of T. forsythensis ATCC 43037, tfsA and tfsB, respectively, following the names in a recent report [Lee, S.-W., Sabet, M., Um, H. S., Yang, L., Kim, H. C. & Zhu, W. (2006). Gene 371, 102-111] were determined. To verify the function of the S-layer proteins, three mutants with tfsA, tfsB, or both deleted were successfully constructed by a PCR-based overlapping method. S-layer proteins were completely lost in the double mutant. The single-deletion mutants appeared to lose one of the 230 and 270 kDa proteins. Thin-section microscopy clearly revealed that the 230 and 270 kDa proteins composed the S-layer. Although the S-layer proteins may be weakly related to haemagglutinating activity, these proteins were highly responsible for adherence to human gingival epithelial cells (Ca9-22) and KB cells. These results suggest that the S-layer proteins in T. forsythensis play an important role in the initiation stage of oral infection including periodontal disease.

A novel Bacteroidetes symbiont is localized in Scaphoideus titanus, the insect vector of Flavescence dorée in Vitis vinifera.

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Characterization of cytoplasmic fibril structures found in gliding cells of Saprospira sp.

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200-300 nm in length. SDS-PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.

Diversity and phylogenetic affiliations of morphologically conspicuous large filamentous bacteria occurring in the pelagic zones of a broad spectrum of freshwater habitats.

Filamentous bacteria with a conspicuous morphology were found in the majority of the bacterioplankton samples from a variety of freshwater habitats that were studied. These heterotrophic filaments typically account for < 1 to 11% of the total number of bacteria. The biovolume of this morphotype can exceed 40% of the biovolume for all bacteria. Surprisingly, we found hardly any data on these morphologically conspicuous filaments in the literature. Mixed cultures containing these filamentous bacteria were established by cultivation and isolation experiments with samples from different freshwater lakes. Nearly full-length 16S rRNA gene sequences were obtained from several mixed cultures and environmental samples from habitats in Europe, Africa, China, Australia, and New Zealand. Phylogenetic analysis of the sequences showed that three groups form a single monophyletic cluster, the SOL cluster, in the family Saprospiraceae. We developed a set of six nested probes for fluorescence in situ hybridization. Of the six probes, one probe was specific for Haliscomenobacter hydrossis, three probes were specific for the three subclusters (each probe was specific for one subcluster), one probe was specific for the entire SOL cluster, and another probe targeted almost the entire Saprospiraceae family. Specific hybridization of environmental samples and enrichments showed that the members of the three subclusters exhibited the same filamentous morphology. So far, using the subcluster-specific probes, we have not been able to detect any bacteria with a differing morphology. We conclude that the SOL cluster bacteria are an integral part of bacterioplankton in many freshwater habitats. They potentially account for a large fraction of the total bacterial biomass but have been underrepresented in molecular diversity studies so far.

FR901451, a novel inhibitor of human leukocyte elastase from Flexibacter sp. I. Producing organism, fermentation, isolation, physico-chemical and biological properties.

A novel human leukocyte elastase (HLE) inhibitor, FR901451 was discovered in the fermentation broth of a bacteria. The bacteria was identified as Flexibacter sp. No. 758. FR901451 has a molecular weight of 1269 and a molecular formula of C60H79N13O18. The mode of inhibition against HLE is competitive, with a Ki value of 9.8 x 10(-9) M.

Flexibacter ovolyticus sp. nov., a pathogen of eggs and larvae of Atlantic halibut, Hippoglossus hippoglossus L.

A psychrotrophic Flexibacter sp., Flexibacter ovolyticus sp. nov., was isolated from the adherent bacterial epiflora of Atlantic halibut (Hippoglossus hippoglossus L.) eggs and was shown to be an opportunistic pathogen for halibut eggs and larvae. The strains which we isolated had the enzymatic capacity to dissolve both the chorion and the zona radiata of the egg shells. A total of 35 isolates were characterized by using morphological and biochemical tests. These strains were rod shaped, gram negative, Kovacs oxidase positive, and pale yellow and exhibited gliding motility. They did not produce acid from any of the wide range of carbohydrates tested. Our isolates had the ability to degrade gelatin, tyrosine, DNA, and Tween 80. Starch, cellulose, and chitin were not degraded. The strains were catalase and nitrate reductase positive, did not produce H2S, and did not grow under anaerobic conditions. F. ovolyticus resembles Flexibacter maritimus, but differs from the latter species in several biochemical and physiological characteristics. DNAs from F. ovolyticus strains had guanine-plus-cytosine contents which ranged from 30.3 to 32.0 mol% (strains EKC001, EKD002T [T = type strain], and VKB004), and DNA-DNA hybridization studies revealed levels of relatedness between F. ovolyticus EKD002T and F. maritimus NCMB 2154T and NCMB 2153 of 42.7 and 30.0%, respectively. Compared with previously described Cytophaga and Flexibacter spp. with low guanine-plus-cytosine contents, F. ovolyticus constitutes a new species. Strain EKD002 (= NCIMB 13127) is the type strain of the new species.