Endobacteria in the tentacles of selected cnidarian species and in the cerata of their nudibranch predators

This is the first genetic analysis comparing cultured endobacteria discovered in the tentacles of cnidarian species (Tubularia indivisa, Tubularia larynx, Corymorpha nutans, Sagartia elegans) with those found in the cerata tips of selected nudibranch species (Berghia caerulescens, Coryphella lineata, Coryphella gracilis, Janolus cristatus, Polycera faeroensis, Polycera quadrilineata, Doto coronata, Dendronotus frondosus). Shared pathogenic activities were found among other microorganisms in the Pseudoalteromonas tetraodonis group (TTX), and the Vibrio splendidus group (haemolytic, septicaemic, necrotic activity). Specific autochthonous endobacteria of extremely low similarity to their next neighbours were detected in nudibranch cerata. These organisms are regarded as new and unknown endobacteria; among them were Pseudoalteromonas luteoviolacea (95%), Orientia tsutsugamushi (84%), Gracilimonas tropica (96%), Balneola alkaliphia (95%), Loktanella rosea (97%). SEM micrographs provide insight into endobacterial aggregates in cnidarian tentacles and nudibranch cerata. Since certain nudibranch predators prey on cnidarian species, it is assumed that cnidarian tentacle bacteria are directly transferred to nudibranch cerata. The pathogenic endobacteria may contribute to the chemical defence of both the nudibranch and cnidarian species investigated.     

Endobacterial morphotypes in nudibranch cerata tips: a SEM analysis

The SEM investigation of nudibranch cerata material exhibits endobacterial morphotypes found in 12 out of 13 species tested: Aeolidia papillosa, Berghia caerulescens, Coryphella brownii, Coryphella lineata, Coryphella verrucosa, Cuthona amoena, Facelina coronata, Flabellina pedata, Dendronotus frondosus, Doto coronata, Tritonia plebeia and Janolus cristatus. Endobacteria could not be detected inside Tritonia hombergi. Endobacterial morphology found inside nudibranch species was compared to bacterial morphotypes detected earlier in tentacles of cnidarian species. SEM micrographs show endobacterial analogy among nudibranch species, but also similarity to cnidarian endobacteria investigated earlier. Of course, morphological data of microbes do not allow their identification. However, since most of these nudibranch species prey on cnidaria, it cannot be excluded that many of the endobacteria detected inside nudibranch species may originate from their cnidarian prey. Our previous data describing genetic affiliation of endobacteria from nudibranchian and cnidarian species support this assumption. Dominant coccoid endobacteria mostly exhibit smooth surface and are tightly packed as aggregates and/or wrapped in envelopes. Such bacterial aggregate type has been described previously in tentacles of the cnidarian species Sagartia elegans. Similar coccoid bacteria, lacking envelopes were also found in other nudibranch species. A different type of coccoid bacteria, characterized by a rough surface, was detected inside cerata of the nudibranch species Berghia caerulescens, and surprisingly, inside tentacles of the cnidarian species Tubularia indivisa. In contrast to cnidarian endobacteria, rod-shaped microorganisms are largely absent in nudibranch cerata.     

Detection of Arcobacter spp. in the Coastal Environment of the Mediterranean Sea

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.     

Taxonomic studies of deep-sea barophilic Shewanella strains and description of Shewanella violacea sp. nov.

Several barophilic Shewanella species have been isolated from deep-sea sediments at depths of 2,485– 6,499 m. From the results of taxonomic studies, all of these isolates have been identified as strains of Shewanella benthica except for strain DSS12. Strain DSS12 is a member of a novel, moderately barophilic Shewanella species isolated from the Ryukyu Trench at a depth of 5,110 m. On Marine Agar 2216 plates, this organism produced a violet pigment, whereas the colonies of other isolates (S. benthica) were rose-colored. Phylogenetic analysis based on 16 S ribosomal RNA gene sequences showed that strain DSS12 represents a separate lineage within the genus Shewanella that is closely related to S. benthica and particularly to the members of the Shewanella barophiles branch. The temperature range for growth and some of the biochemical characteristics indicate that strain DSS12 differs from other Shewanella species. Furthermore, strain DSS12 displayed a low level of DNA similarity to the Shewanella type strains. Based on these differences, it is proposed that strain DSS12 represents a new deep-sea Shewanella species. The name Shewanella violacea (JCM 10179) is proposed. Received: 15 May 1998 / Accepted: 15 July 1998     

The presence and distribution of Antho-RFamide-like material in scyphomedusae

Summary The nervous systems of the scyphomedusae Chrysaora hysoscella, Cyanea capillata and Cyanea lamarckii (Phylum Cnidaria) were stained using an antiserum against the anthozoan neuropeptide Antho-RFamide. Staining was widespread in all three species. In Chrysaora, the antiserum revealed nerve nets in the subumrella and exumbrella ectoderm, in both faces of the oral lobes, and in the endoderm lining the subumbrella and exumbrella surfaces of the gastric cavity. The most prominent staining occurred in a dense plexus of neurons in the ectoderm at the base of the tentacles. This nerve net sent projections into the subumbrella ectoderm. For the most part, staining in the two species of Cyanea was similar to that in Chrysaora, with a few exceptions. These include the presence, in Cyanea, of an obvious tentacular nerve tract and nerve nets associated with clusters of cnidocytes in the tentacles. Radioimmunoassays of extracts from Chrysaora and Cyanea lamarkii revealed that both species contain large amounts of Antho-RFamide-like material (up to 55 nmol/animal). The results indicate that Antho-RFamide-like neuropeptides are widespread in scyphomedusae.     

Antimicrobial resistance and virulence genes profiles of Arcobacter butzleri strains isolated from back yard chickens and retail poultry meat in Chile

This research aims to investigate the presence and pathogenic potential of Arcobacter in poultry meat samples purchased in the retail market of Valdivia (South of Chile) as well as in faecal samples from backyard chickens from rural areas around this city. The isolates obtained were identified by molecular methods. Furthermore, putative virulence genes were assessed by PCR and the antimicrobial resistance was tested by phenotypic methods. Arcobacter was present in 41·6% of the samples, with the highest value in retail poultry meat (55·7%) followed by backyard production (28·0%). Arcobacter butzleri was the most prevalent species (75·6%) followed by Arcobacter skirrowii (14·8%) and Arcobacter cryaerophilus (9·6%). An 8·5% of A. butzleri strains from meat were resistant to both ciprofloxacin and tetracycline and 6·1% were resistant to erythromycin, while none was resistant to gentamycin, unlike strains from domestic chickens, which showed no resistance. Furthermore, A. butzleri strains from chicken meat presented a higher prevalence of virulence genes than strains from domestic chickens. In fact, in this last group, some genes (hecA, hecB and irgA) were completely absent. Therefore, this study provides insight on the epidemiology of Arcobacter in Chilean poultry and suggests that under traditional breeding conditions strains are, apparently, less pathogenic and drug resistant.     

Difference in NaCl tolerance of membrane-bound 5′-nucleotidases purified from deep-sea and brackish water Shewanella species

Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5′-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl₂ for their activities, and they exhibited the same pH dependency and substrate specificity for 5′-nucleotides. However, S. violacea 5′-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5′-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5′-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.

     

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>MR-1

Background  

EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

Cytologic and Genetic Characteristics of Endobiotic Bacteria and Kleptoplasts of Virgulinella fragilis (Foraminifera)

The benthic foraminifer Virgulinella fragilis Grindell and Collen 1976 has multiple putative symbioses with both bacterial and kleptoplast endobionts, possibly aiding its survival in environments from dysoxia (5–45 μmol‐O₂/L) to microxia (0–5 μmol‐O₂/L) and in the dark. To clarify the origin and function of V. fragilis endobionts, we used genetic analyses and transmission electron microscope observations. Virgulinella fragilis retained δ‐proteobacteria concentrated at its cell periphery just beneath the cell membranes. Unlike another foraminifer Stainforthia spp., which retains many bacterial species, V. fragilis has a less variable bacterial community. This suggests that V. fragilis maintains a specific intracellular bacterial flora. Unlike the endobiotic bacteria, V. fragilis klepto‐plasts originated from various diatom species and are found in the interior cytoplasm. We found evidence of both retention and digestion of kleptoplasts, and of fragmentation of the kleptoplastid outer membrane that likely facilitates transport of kleptoplastid products to the host. Accumulations of mitochondria were observed encircling endobiotic bacteria. It is likely that the bacteria use host organic material for carbon oxidation. The mitochondria may use oxygen available around the δ‐proteobacteria and synthesize adenosine triphosphate, perhaps for sulfide oxidation.     

Molecular Analysis of Microbiota Along the Digestive Tract of Juvenile Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Dominant bacterial microbiota of the gut of juvenile farmed Atlantic salmon was investigated using a combination of molecular approaches. Bacterial community composition from the stomach, the pyloric caeca, and the intestine was assessed by extracting DNA directly from each gut compartment. Temporal temperature gradient gel electrophoresis (TTGE) analysis of 16S ribosomal DNA (rDNA) amplicons showed very similar bacterial compositions throughout the digestive tract. Band sequencing revealed a narrow diversity of species with a dominance of Pseudomonas in the three compartments. However, cloning revealed more diversity among the Pseudomonas sequences. To confirm these results, we analyzed the bacterial community by amplifying the variable 16S–23S rDNA intergenic spacer region (ITS). Similar ITS profiles were observed among gastrointestinal compartments of salmon, confirming the TTGE results. Moreover, the dominant ITS band at 650 bp, identified as Pseudomonas, was observed in the ITS profile from fish collected in two seasons (July 2003 and 2004). In contrast, aerobic culture analysis revealed Shewanella spp. as the most prevalent isolate. This discrepancy was resolved by evaluating 16S rDNA and ITS polymerase chain reaction amplification efficiency from both Shewanella and Pseudomonas isolates. Very similar efficiencies were observed in the two bacteria. Hence, this discrepancy may be explained by preferential cultivation of Shewanella spp. under the experimental conditions. Also, we included analyses of pelleted feed and the water influent to explore environmental influences on the bacterial composition of the gut microbiota. Overall, these results indicate a homogeneous composition of the bacterial community composition along the gastrointestinal tract of reared juvenile salmon. This community is mainly composed of Pseudomonas spp., which could be derived from water influent and may be selectively associated with salmon in this hatchery.     

Structural and Developmental Disparity in the Tentacles of the Moon Jellyfish Aurelia sp.1

Tentacles armed with stinging cells (cnidocytes) are a defining trait of the cnidarians, a phylum that includes sea anemones, corals, jellyfish, and hydras. While cnidarian tentacles are generally characterized as structures evolved for feeding and defense, significant variation exists between the tentacles of different species, and within the same species across different life stages and/or body regions. Such diversity suggests cryptic distinctions exist in tentacle function. In this paper, we use confocal and transmission electron microscopy to contrast the structure and development of tentacles in the moon jellyfish, Aurelia species 1. We show that polyp oral tentacles and medusa marginal tentacles display markedly different cellular and muscular architecture, as well as distinct patterns of cellular proliferation during growth. Many structural differences between these tentacle types may reflect biomechanical solutions to different feeding strategies, although further work would be required for a precise mechanistic understanding. However, differences in cell proliferation dynamics suggests that the two tentacle forms lack a conserved mechanism of development, challenging the textbook-notion that cnidarian tentacles can be homologized into a conserved bauplan.     

Metabolic flux analysis of Shewanella spp. reveals evolutionary robustness in central carbon metabolism

Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and freshwater environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal medium with [3‐¹³C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23–0.29 h^?1) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo‐steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance of metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636–640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate. Biotechnol. Bioeng. 2009;102: 1161–1169. © 2008 Wiley Periodicals, Inc.     

First multi-locus sequence typing scheme for Arcobacter spp.

Background  

Arcobacter spp. are a common contaminant of food and water, and some species, primarily A. butzleri and A. cryaerophilus, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first Arcobacter multilocus sequence typing (MLST) method for A. butzleri, A. cryaerophilus, A. skirrowii, A. cibarius and A. thereius.     

Moritella japonica sp. nov., a novel barophilic bacterium isolated from a Japan Trench sediment

Strain DSK1 is a novel moderately barophilic bacterium isolated from the Japan Trench at a depth of 6,356 m. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that strain DSK1 represents a separate lineage with the Shewanella barophiles branch and is closely related to Moritella marina. Comparisons of the temperature and pressure range for growth and some biochemical characteristics indicate that strain DSK1 differs from M. marina and Shewanella barophilic species. Furthermore, strain DSK1 displays a low level of DNA similarity to the Moritella and Shewanella type strains; however this isolate characteristically produces DHA (22:6) as a membrane fatty acids, and the fatty acid profile of this strain is similar to that of M. marina. Because of these differences, strain DSK1 appears to represent a novel deep-sea Moritella species. The name Moritella japonica is proposed. The type strain is JCM 10249.     

希瓦氏菌生物被膜研究进展

希瓦氏菌（Shewanella spp.）是海产品中常见的优势腐败菌,易在食品加工设备表面形成生物被膜而难以清除。生物被膜的存在不仅会造成食品的持续污染和交叉污染,也会影响加工设备的使用,从而对国民健康和经济发展造成威胁。目前,针对希瓦氏菌生物被膜的研究主要集中在表型研究上,对其生物被膜形成分子机制的研究尚处于起步阶段。总结希瓦氏菌生物被膜的形成过程,重点论述希瓦氏菌生物被膜的形成机制并对希瓦氏菌生物被膜控制方法进行简要概括,展望未来的研究方向,以期为希瓦氏菌生物被膜的深入研究提供参考。     

Characterization of the lipids of psychrophilic bacteria <Emphasis Type="Italic">Shewanella frigidimarina</Emphasis> isolated from sea ice of the Sea of Japan

Lipids of ten Shewanella frigidimarina strains isolated from sea ice samples of coastal areas of the Sea of Japan and of the type strains of psychrophilic bacteria S. frigidimarina ACAM 591^T and S. hanedai JCM 20706^T were analyzed. Most of the new isolates contained isoprenoid quinones typical of the genus Shewanella (Q-7, Q-8, MK-7, and MMK-7), a high level of branched acids (i-13:0 and i-15:0), and polyunsaturated fatty acid (20:5 ω3). Phospholipid fractions of marine isolates and the type strain S. frigidimarina ACAM 591^T contained not only the main phospholipids (phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol), but also an unknown phosphoaminolipid, which is probably typical of this bacterial species. The isolates exhibited a high level of phylogenetic similarity but were phenotypically heterogeneous. Two strains distinguished by their phenotypic characteristics differed also in the composition of fatty acids, isoprenoid quinones, and phospholipids. The use of chemotaxonomic markers for primary species identification of psychrophilic bacteria of the genus Shewanella is discussed.     

The Microbiota of Freshwater Fish and Freshwater Niches Contain Omega-3 Fatty Acid-Producing Shewanella Species

Approximately 30 years ago, it was discovered that free-living bacteria isolated from cold ocean depths could produce polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) (20:5n-3) or docosahexaenoic acid (DHA) (22:6n-3), two PUFA essential for human health. Numerous laboratories have also discovered that EPA- and/or DHA-producing bacteria, many of them members of the Shewanella genus, could be isolated from the intestinal tracts of omega-3 fatty acid-rich marine fish. If bacteria contribute omega-3 fatty acids to the host fish in general or if they assist some bacterial species in adaptation to cold, then cold freshwater fish or habitats should also harbor these producers. Thus, we undertook a study to see if these niches also contained omega-3 fatty acid producers. We were successful in isolating and characterizing unique EPA-producing strains of Shewanella from three strictly freshwater native fish species, i.e., lake whitefish (Coregonus clupeaformis), lean lake trout (Salvelinus namaycush), and walleye (Sander vitreus), and from two other freshwater nonnative fish, i.e., coho salmon (Oncorhynchus kisutch) and seeforellen brown trout (Salmo trutta). We were also able to isolate four unique free-living strains of EPA-producing Shewanella from freshwater habitats. Phylogenetic and phenotypic analyses suggest that one producer is clearly a member of the Shewanella morhuae species and another is sister to members of the marine PUFA-producing Shewanella baltica species. However, the remaining isolates have more ambiguous relationships, sharing a common ancestor with non-PUFA-producing Shewanella putrefaciens isolates rather than marine S. baltica isolates despite having a phenotype more consistent with S. baltica strains.     

Conserved synteny at the protein family level reveals genes underlying Shewanella species�� cold tolerance and predicts their novel phenotypes

Bacteria of the genus Shewanella can thrive in different environments and demonstrate significant variability in their metabolic and ecophysiological capabilities including cold and salt tolerance. Genomic characteristics underlying this variability across species are largely unknown. In this study, we address the problem by a comparison of the physiological, metabolic, and genomic characteristics of 19 sequenced Shewanella species. We have employed two novel approaches based on association of a phenotypic trait with the number of the trait-specific protein families (Pfam domains) and on the conservation of synteny (order in the genome) of the trait-related genes. Our first approach is top-down and involves experimental evaluation and quantification of the species?? cold tolerance followed by identification of the correlated Pfam domains and genes with a conserved synteny. The second, a bottom-up approach, predicts novel phenotypes of the species by calculating profiles of each Pfam domain among their genomes and following pair-wise correlation of the profiles and their network clustering. Using the first approach, we find a link between cold and salt tolerance of the species and the presence in the genome of a Na⁺/H⁺ antiporter gene cluster. Other cold-tolerance-related genes include peptidases, chemotaxis sensory transducer proteins, a cysteine exporter, and helicases. Using the bottom-up approach, we found several novel phenotypes in the newly sequenced Shewanella species, including degradation of aromatic compounds by an aerobic hybrid pathway in Shewanella woodyi, degradation of ethanolamine by Shewanella benthica, and propanediol degradation by Shewanella putrefaciens CN32 and Shewanella sp. W3-18-1.